Devices, compositions and methods for imaging with raman scattering

ABSTRACT

Methods, systems and computer-accessible medium for imaging a living cell or a living organism with bond-edited compounds using stimulated Raman scattering are disclosed. The method comprises the steps of introducing one or more bond-edited compounds into a live cell or a living organism, and detecting a vibrational tag in the cell or organism with stimulated Raman scattering. Also disclosed are methods for detecting a disease condition in a subject, methods for monitoring treatment for a disease condition, methods for screening an agent, methods for tracing a cellular process in a live cell using bond-edited compounds in combination with stimulated Raman scattering. Also disclosed are a composition for labeling a target cell with at least one bond-edited compound and devices for imaging bond-edited compounds by stimulated Raman scattering.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. Non Provisional patentapplication Ser. No. 14/974,992, filed on Dec. 18, 2015, which issues asU.S. Pat. No. 11,408,892 on Aug. 9, 2022, which is acontinuation-in-part of International Patent Application No.PCT/US2014/042936, filed on Jun. 18, 2014 which claims the priority ofU.S. Provisional Application Ser. No. 61/836,235, filed Jun. 18, 2013,and 61/946,296, filed Feb. 28, 2014. This application also relates toand claims priority from U.S. Patent Application No. 62/112,906, filedon Feb. 6, 2015. The entire disclosures of the above applications areincorporated herein by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under EB016573 and_EB020892, awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE DISCLOSURE

The present disclosure relates generally to imaging technology, and inparticular to vibrational microscopy and spectroscopy with Ramanscattering technology.

BACKGROUND INFORMATION

Innovations in light microscopy have expanded the knowledge ofbiological processes at the microscopic level. In particular,fluorescence microscopy, utilizing versatile fluorescent probes (e.g.genetic labeling of fluorescent proteins, organic dyes and quantum dots)(See, e.g., References 1-3), can facilitate specific detection ofmolecules of interest in biological systems, facilitating people toactually visualize and understand fundamental processes. Takingadvantage of the development of fluorescent probes (e.g. brighter, morephotostable, multicolor etc.) (See, e.g., Reference 4), fluorescentmicroscopy such as confocal microscopy, two-photon microscopy, singlemolecule microscopy and superresolution microscopy have enableddetection of structures that can be much deeper and finer than before.However, according to quantum mechanics (e.g. particle in the box), thechromophore within a fluorophore have to be a large conjugation systemin order for the efficient absorption in the visible spectrum. Thus, inspite of the significance in various applications such as in cellbiology, fluorescent tags intrinsically cannot be properly used fortagging small molecules such as glucose, nucleosides, amino acids,choline, fatty acids and small molecule drugs, for their relativelylarge size perturbs with the small molecule dynamics.

An opposite strategy for visualizing these important building blocksmall molecules in biological systems can be label-free imaging.Representative imaging procedures of the kind can include vibrationmicroscopies based on infrared absorption and Raman scattering detectingthe characteristic vibrational mode of specific chemical bond from themolecules themselves (See, e.g., References 5-9). Other label-freeprocedures can be second harmonic generation (“SHG”), imaging specialnon-centrosymmetric structures, third harmonic generation (“THG”),sensing interfaces and optical heterogeneities and optical coherencetomography (“OCT”), measuring the backscattered light from tissuesthrough low-coherence interferometry. However, label-free imaging cansuffer from two fundamental problems: first, there can be insufficientspecificity because small molecules usually do not have uniquespectroscopic signature in the vast pool of other biomolecules; second,there can be unsatisfying sensitivity due to usually low concentrationof the small molecules in the biological systems.

As an imaging tag, alkyne (e.g. carbon-carbon triple bond) can offerthree advantages over others. First, alkyne is only a chemical bond,second alkyne can enable background-free detection, and third, alkynecan be inert to react with any intrinsic bio-molecules in the biologicalsystems. In fact, alkyne can be widely used in the powerfulbioorthogonal chemistry utilizing alkyne-azide specific click-chemistryreaction for various purposes (See, e.g., References 10-12). Forexample, using alkyne tagged molecule of interest followed by azidetagged detection reagent (e.g. affinity probes or fluorescent tag) canenable detection using mass spectrometry or fluorescence microscopy.

The proteome of a cell can be highly dynamic in nature and tightlyregulated by both protein synthesis and degradation to actively maintainhomeostasis. Many intricate biological processes, such as cell growth,differentiation, diseases and response to environment stimuli, canrequire protein synthesis and translational control (See, e.g.,Reference 24). In particular, long-lasting forms of synaptic plasticity,such as those underlying long-term memory, can need new proteinsynthesis in a space- and time-dependent manner (See, e.g., References26-30). Therefore, direct visualization and quantification of newlysynthesized proteins at a global level can be indispensable tounraveling the spatial-temporal characteristics of the proteomes in livecells.

Extensive efforts have been devoted to probing protein synthesis viafluorescence contrast. The inherent fluorescence of green fluorescentprotein (“GFP”) and its genetic encodability, can the following of agiven protein of interest inside living cells with high spatial andtemporal resolution (See, e.g., References 29 and 30). However, GFPtagging through genetic manipulation works only on individual proteins,and not at the whole proteome level. To probe newly synthesized proteinsat the proteome level, a powerful procedure named bioorthogonalnoncanonical amino acid tagging (BONCAT) was developed by metabolicincorporation of unnatural amino acids containing reactive chemicalgroups such as azide or alkyne. (See, e.g., References 31-37). A relatedlabeling method was recently demonstrated using an alkyne analog ofpuromycin. (See, e.g., Reference 28). Newly synthesized proteins canthen be visualized through subsequent conjugation of the reactive aminoacids to fluorescent tags via click chemistry. (See, e.g., Reference29). Unfortunately, these fluorescence-based methods generally usenon-physiological fixation and subsequent dye staining and washing.

In addition to fluorescence tagging, radioisotope or stable isotope,labeling can be another powerful tool to trace and quantify proteomedynamics. Classical radioisotope-labeled amino acids (e.g.,35S-methionine) can provide vigorous analysis of global proteinsynthesis. However, samples must be fixed and then exposed to film forautoradiography. For stable isotopes, the discovery of deuterium by Ureyin 1932 immediately led to the pioneer work of Schoenheimer andRittenberg studying intermediary metabolism. (See, e.g., References 40and 51). To study proteome changes between different cells or underdifferent conditions, stable isotope labeling by amino acids in cellculture (“SILAC”) coupled with mass spectrometry (“MS”) has matured intoa popular method for quantitative proteomics (See, e.g., References42-45). However, SILAC-MS does not usually provide spatial informationdown to sub-cellular level and its invasive nature limits itsapplication for live cell imaging. The same limitation applies to therecent ribosome profiling study using deep sequencing procedure (See,e.g., Reference 46).

Spontaneous Raman microscopy has been used for label-free molecular andbiomedical imaging (See, e.g., References 8, 13, 17, 59 and 73-77).However, this technology suffers from low sensitivity and slow imagingspeed.

Among various optical imaging techniques, fluorescence microscopy may beone of the most widely adopted imaging modalities, because it offerssingle-molecule sensitivity for the visualization of a wide variety ofmolecules labeled with fluorophores. (See, e.g., References 79-81). Suchsensitivity, together with recent technical developments, has enabledthe use of a two-photon fluorescence microscopy for deep tissue and invivo imaging (see, e.g., Reference 82), super-resolution fluorescencemicroscopy that breaks the diffraction limit for nanometer scaleresolution (see, e.g., References 83-85), and fluorescence resonanceenergy transfer microscopy for imaging intracellular molecularinteractions. (See, e.g., Reference 86). However, fluorescencemicroscopy generally probes the electronic transition of thefluorophores, resulting in both featureless and broadband (e.g.,bandwidth of about 50-100 nm) absorption and emission spectra, mainlydue to strong electronic state dephasing. (See, e.g., References 87 and88). Thus, two fluorophores with distinct structures can result inoverlapping and unresolvable spectra, which likely limits thesimultaneously detected fluorophores (e.g., typically to 4).

Currently, non-fluorescence based imaging techniques offeringsingle-molecule sensitivity are commonly absorption-based methods, suchas measuring photothermal contrast, ground state depletion from a singlemolecule, or using balanced detector and index-matched sample geometry(see, e.g., References 89-91), which can all yield a similar number ofcolors for simultaneous multiplex imaging as in fluorescence microscopy.Nevertheless, multicolor imaging of up to tens of colors can be highlydemanded for real biomedical applications, such as imaging various typesof tumor receptors simultaneously in cancer research (see, e.g.,References 92 and 93), to detect cancer markers in biomedicaldiagnostics by flow cytometer (see, e.g., Reference 94), and to followthe highly dynamic focal adhesion complex for the research of cellinteractions with the extracellular matrix. (See, e.g., Reference 95).

As an alternative, Raman microscopy can potentially image up to tens orhundreds of molecules simultaneously by probing the vibrationaltransition of the molecules and offering distinct and sharp Raman peakswith chemical specificity. (See, e.g., Reference 96). Thus, twomolecules with close chemical structures could possibly be resolved inRaman spectrum. However, spontaneous Raman, as a single-laser technique,likely suffers from an extremely weak sensitivity that can be about1010-1012 times weaker than fluorescence. Thus, for imaging biologicalsamples, spontaneous Raman microscopy can be an undesirable techniquebecause of the long acquisition time needed, and the large sampleauto-fluorescence background. A current Raman technique, SurfacedEnhanced Raman Scattering (“SERS”), provides a remarkable sensitivityeven at single molecule level (see, e.g., References 97 and 98).However, this technique relies on the enhancement from metal surfaceplasmons that can benefit from nanometer-precision positioning betweenthe sample and the metal surface, therefore prohibiting its applicationin intracellular cell imaging.

Thus, it may be beneficial to have an imaging strategy that makes up forthe gap between fluorescence microscopy and label-free imaging for thesensitive and specific detection of small molecules while offeringminimum perturbation to the biological systems (e.g. to have small tagswith distinct spectroscopic characteristics), and which can overcome atleast some of the deficiencies described herein above.

SUMMARY OF EXEMPLARY EMBODIMENTS

One exemplary aspect of the present disclosure relates to a method forobtaining biological information in a living cell or a living organismwith bond-edited compounds using stimulated Raman scattering. The methodcomprises the steps of introducing one or more bond-edited compoundsinto a live cell or a living organism, and detecting a vibrational tagin the cell or organism with stimulated Raman scattering.

Another exemplary aspect of the present disclosure relates to methodsfor making a bond-edited compound.

Another exemplary aspect of the present disclosure relates to a methodfor detecting a disease condition in a subject, comprising:administering to said subject a composition comprising a bond-editedcompound targeting a disease tissue or pathogen, and detecting saidbond-edited compound by stimulated Raman scattering.

Another exemplary aspect of the present disclosure relates to a methodfor monitoring a treatment for a disease condition. The method comprisesadministering to the subject a composition comprising a bond-editedcompound and detecting the bond-edited compound by stimulated Ramanscattering at a first time point, performing the treatment after thefirst time point, further administering to said subject the compositioncomprising a bond-edited compound, and detecting the bond-editedcompound by stimulated Raman scattering at a second time point, andcomparing images obtained at the two time points,

Another exemplary aspect of the present disclosure relates to a methodfor screening an agent. The method comprises administering the agent andat least one bond-edited compound to a live cell or organism, detectingthe bond-edited compound in the live cell or organism using stimulatedRaman scattering, and selecting a candidate agent based on one or morepredetermined criteria, such as the uptake, accumulation, trafficking ordegradation of the bond-edited compound by the said live cell ororganism.

Another exemplary aspect of the present disclosure relates to a devicefor imaging bond-edited compounds by stimulated Raman scattering. Thedevice comprises a first single-wavelength laser source that produces apulse laser beam of a first wavelength, a second single-wavelength lasersource that produces a pulse laser beam of a second wavelength, amodulator that modulates the pulse laser beam of one of the first orsecond laser source, a photodetector that is capable of or configured todetect stimulated Raman scattering from a biosample, and a computer.

Another exemplary aspect of the present disclosure relates to anon-transitory computer-accessible medium having stored thereoncomputer-executable instructions for determining data associated with atleast one tissue, wherein, when a computer hardware arrangement executesthe instructions, the computer arrangement is configured to performprocedures comprising: receiving first information related to at leastone bond between at least two atoms attached to a metabolite; anddetermining the data based on the at least one bond.

Another exemplary aspect of the present disclosure relates to a methodfor determining data associated with at least one tissue, comprising:receiving first information related to at least one bond between atleast two atoms attached to a metabolite; and using a computer hardwarearrangement, determining the data based on the at least one bond.

Another exemplary aspect of the present disclosure relates to a systemfor determining data associated with at least one tissue, comprising: acomputer processing arrangement configured to receive first informationrelated to at least one bond between at least two atoms attached to ametabolite; and determine the data based on the at least one bond.

Another exemplary aspect of the present disclosure relates to apre-mixed essential amino acid combination, comprising: at least onenon-deuterated essential amino acid; and at least 5 deuterated essentialamino acids.

Another exemplary aspect of the present disclosure relates to a methodfor exciting a light absorbing molecule(s) can include, for example,labeling a target molecule(s) to create the light absorbing molecule(s)using a label(s) having a resonance energy level, and forwarding aradiation(s) to the light absorbing molecule(s) at an energy outputlevel so as to excite the light absorbing molecule(s), where adifference between the resonance energy level and the energy outputlevel can be within a predetermined range. The range can be betweenabout 500 cm-1 to about 2000 cm-1. The radiation(s) can include a laserlight, and can also include two radiations. The radiation(s) can begenerated using a stimulated Raman scattering arrangement. The label(s)can include a chromophore(s).

In certain exemplary embodiments of the present disclosure, thechromophore(s) can include a dye, which can include an alkyne(s). Thealkyne(s) can include an isotopically modified alkyne(s). An image(s)can be generated using a resultant radiation received from the excitedlight absorbing molecule(s) that can be based on the forwardedradiation(s). The light absorbing molecule(s) can include a chromophoreor a fluorophore.

In some exemplary embodiments of the present disclosure, a furthertarget molecule(s) can be labeled to create a further light absorbingmolecule(s) using a further label(s) having a further resonance energylevel, and the radiation(s) can be forwarded to the further lightabsorbing molecule(s) at the energy output level so as to excite thatfurther light absorbing molecule(s), where the difference between thefurther resonance energy level and the energy output level can be withinthe predetermined range. The further resonance energy level can bedifferent than the resonance energy level. A vibrational spectrum of thelight absorbing molecule(s) can be different than the vibrationalspectrum of the further light absorbing molecule(s).

Another exemplary aspect of the present disclosure relates to a system,which can include, for example, a label(s) of a target molecule(s)having a resonance energy level, and a radiation generatingarrangement(s) providing a radiation(s) to the target molecule(s) thatcan have an energy output level, where a difference between theresonance energy level and the energy output level can be within apredetermined range. The predetermined range can be between about 500cm-1 to about 2000 cm-1. The radiation generating arrangement(s) caninclude a stimulated Raman scattering arrangement. The label can includea chromophore(s), which can include a dye.

Still a further exemplary embodiment of the present disclosure can be alabel, which can include, for example, a chromophore(s), and anisotopically modified alkyne(s). The label can also include achemical(s) or a light absorbing protein(s).

A further exemplary embodiment of the present disclosure is a method forimaging a living cell or a living organism, which can includeintroducing an effective amount of a bond-edited compound into a livecell or a living organism, where the bond-edited compound comprises avibrational tag, and detecting the vibrational tag in the cell or theorganism with stimulated Raman scattering (SRS) imaging. The bond-editedcompound can be a small molecule The bond-edited compound can includeone, two, three, four, five, six, seven, eight, nine, ten or morevibrational tags. The vibrational tags can be the same type of tags or amixture of one or more different tags, and can include an alkyne tag, anazide tag, an isotope label, or a combination of an alkyne tag and acarbon-deuterium bond tag. The isotope label can be a carbon-deuteriumbond tag. The bond-edited compound can include a vibrational tag(s) of—C≡C—, —C≡N, —N═N═N, —C≡C—C≡C—, —C≡C—C≡N, —C-D, and —C≡C-D, at least one13C atom or one deuterium atom, an amino acid, a nucleoside or anucleotide, a fatty acid, a monosaccharide or a disaccharide, glucose, aglucose derivative or propargyl glucose, or a cytokine or chemokine.

In some exemplary embodiments of the present disclosure, the amino acidcan be an essential amino acid, and can be histidine, isoleucine,leucine, lysing, methionine, phenylalanine, threonine, tryptophan orvaline. The bond-edited compound can also be selected from anti-canceragents, anti-inflammatory agents, anti-bacterial agents, anti-fungalagents or anti-viral agents. The vibrational tag can be transferred fromthe bond-edited compound to a down-stream metabolite of the bond-editedcompound, and can be detected in the down-stream metabolite.

In yet another exemplary embodiment of the present disclosure is amethod for imaging a living cell or a living organism, which can includeintroducing into the live cell or organism a mixture of two or morebond-edited compounds wherein the two or more bond-edited compounds eachcomprises a different vibrational tag, and imaging with stimulated Ramanscattering at two or more different wavelengths to detect thevibrational tag on each of the two or more bond-edited compounds. Thetwo or more bond-edited compounds can include EU-13C2, EdU-13C and17-ODYA, and can different cellular components, the same cellularcomponent but at different time period, different types of cells in theliving organism, or two or more bond-edited compounds detected using alinear combination algorithm.

In certain exemplary embodiments of the present disclosure, thevibrational tag on each of the two or more bond-edited compounds can bedetected using a linear combination algorithm.

Another exemplary embodiment of the present disclosure can be a methodfor making a alkyne-tagged compound, which can include adding propargylbromide to a compound of formula S1 in the presence of DMF and sodiumhydride to produce a compound of formula S2;

and, adding water and an ion exchange resin to the compound of formulaS2 to produce a compound of formula S3

Still a further exemplary embodiment of the present disclosure is amethod for making a 13C-tagged compound, which can include reacting acompound of formula 5 with K2CO3, MeOH and H2O to produce the compoundof formula 3:

Yet a further exemplary embodiment of the present disclosure is a methodfor making a 13C-tagged compound, which can include reacting a compoundof formula 10 with TBAF, K2CO3, MeOH and H2O to produce the compound offormula 2:

Yet an even further exemplary embodiment of the present disclosure is amethod for making a 13C-tagged compound, comprising: reacting a compoundof formula S6 with K2CO3, MeOH and H2O to produce the compound offormula 13:

An even further exemplary embodiment of the present disclosure is aalkyne-tagged

compound of formula S3,

A compound of formula 13 can be

An even further exemplary embodiment of the present disclosure is amethod for detecting a disease condition in a subject, which can includeadministering to the subject a composition comprising a bond-editedcompound targeting a disease tissue or pathogen, where the bond-editedcompound comprises a vibrational tag, and detecting the vibrational tagby stimulated Raman scattering imaging. The disease condition caninclude cancer, metabolic syndrome, neurodegenerative diseases,inflammatory diseases and microbial infections. The vibrational tag canbe transferred from the bond-edited compound to a down-stream metaboliteof the bond-edited compound, and can be detected in the down-streammetabolite.

Additionally, a method for monitoring a treatment for a diseasecondition in a subject, can include administering to the subject acomposition comprising a bond-edited compound and detecting thebond-edited compound by stimulated Raman scattering imaging at a firsttime point, further administering to the subject the compositioncomprising a bond-edited compound, detecting the bond-edited compound bystimulated Raman scattering imaging at a second time point and comparingimages obtained at the two time points. The first time point can be atime point that can be about or prior to the initiation of the treatmentand the second time point can be a time point that can be after theinitiation of the treatment. The first time point and the second timepoint can be two time points during the course of the treatment.

A further exemplary embodiment of the present disclosure can include amethod for screening a candidate agent, which can include administeringthe candidate agent and a bond-edited compound(s) to a live cell ororganism, detecting the bond-edited compound in the live cell ororganism using stimulated Raman scattering imaging and determining aneffectiveness of the candidate agent based on one or more predeterminedcriteria selected from the group consisting of the uptake, accumulation,trafficking and degradation of the bond-edited compound in the live cellor organism. The candidate agent can be an anti-cancer drug, or a skinregenerating agent.

A still further exemplary embodiment of the present disclosure caninclude a device for imaging bond-edited compounds by stimulated Ramanscattering, which can include a first laser generator that can produce apulse laser beam of a first fixed wavelength, a second laser generatorthat can produce a pulse laser beam of a second fixed wavelength, amodulator that can modulate the pulse laser beam of the first or secondlaser generator, a photodetector that can be adapted to detecting thestimulated Raman scattering from a biosample and a computer whichgenerates an image(s) of the bond-edited compounds based on the detectedstimulated Raman scattering. The first and second laser generators canbe configured to provide a pump radiation and a stokes radiation, eachat a fixed wavelength whose energy difference can be between about 2000and 2500 wavenumbers.

An even further exemplary embodiment of the present disclosure caninclude a system, method and computer-accessible medium for receivingfirst information related to a bond(s) between at least two atomsattached to a metabolite and determining the data based on the bond(s).The bond can be a carbon deuterium bond, a triple carbon bond, a triplecarbon nitrogen bond, or an azide triple nitrogen bond. A deuterium tohydrogen ratio of the tissue can be at least 1 to 5,000. A deuterium tohydrogen ratio of the tissue can be at least 1 to 1,000. A deuterium tohydrogen ratio of the tissue can be at least 1 to at most 100. The datacan include a location of the bond(s). The data can be determined basedon an amplitude of a signal of the bond(s). The data can be determinedusing a stimulated Raman microscopy arrangement, a coherent anti-StokesRaman scattering arrangement, an infrared absorption arrangement, astimulated Raman excited photothermal arrangement, or a stimulated Ramanexcited photoacoustic arrangement.

In certain exemplary embodiments of the present disclosure, a laser ofthe stimulated Raman microscopy arrangement can be tuned to a particularfrequency based on the bond(s). The tissue(s) can include a live animalcell(s). The metabolite can include (i) a deoxyribonucleoside(s), (ii) aribonucleoside(s), (iii) an amino acid(s), (iv) choline, (v) a fattyacid(s), (vi) an Adenosine triphosphate(s), (vii) cholesterol, or (viii)a chemical drug(s).

A pre-mixed essential amino acid combination can include anon-deuterated essential amino acid(s) and at least 3 or at least 4deuterated essential amino acids.

An exemplary method for exciting a light absorbing molecule(s) caninclude, for example, labeling a target molecule(s) to create the lightabsorbing molecule(s) using a label(s) having a resonance energy level,and forwarding a radiation(s) to the light absorbing molecule(s) at anenergy output level so as to excite the light absorbing molecule(s),where a difference between the resonance energy level and the energyoutput level can be within a predetermined range. The range can bebetween about 500 cm-1 to about 2000 cm-1. The radiation(s) can includea laser light, and can also include two radiations. The radiation(s) canbe generated using a stimulated Raman scattering arrangement. Thelabel(s) can include a chromophore(s).

In certain exemplary embodiments of the present disclosure, thechromophore(s) can include a dye, which can include an alkyne(s). Thealkyne(s) can include an isotopically modified alkyne(s). An image(s)can be generated using a resultant radiation received from the excitedlight absorbing molecule(s) that can be based on the forwardedradiation(s). The light absorbing molecule(s) can include a chromophoreor a fluorophore.

In some exemplary embodiments of the present disclosure, a furthertarget molecule(s) can be labeled to create a further light absorbingmolecule(s) using a further label(s) having a further resonance energylevel, and the radiation(s) can be forwarded to the further lightabsorbing molecule(s) at the energy output level so as to excite thatfurther light absorbing molecule(s), where the difference between thefurther resonance energy level and the energy output level can be withinthe predetermined range. The further resonance energy level can bedifferent than the resonance energy level. A vibrational spectrum of thelight absorbing molecule(s) can be different than the vibrationalspectrum of the further light absorbing molecule(s).

Another exemplary embodiment of the present disclosure can be a system,which can include, for example, a label(s) of a target molecule(s)having a resonance energy level, and a radiation generatingarrangement(s) providing a radiation(s) to the target molecule(s) thatcan have an energy output level, where a difference between theresonance energy level and the energy output level can be within apredetermined range. The predetermined range can be between about 500cm-1 to about 2000 cm-1. The radiation generating arrangement(s) caninclude a stimulated Raman scattering arrangement. The label can includea chromophore(s), which can include a dye.

Still a further exemplary embodiment of the present disclosure can be alabel, which can include, for example, a chromophore(s), and anisotopically modified alkyne(s). The label can also include achemical(s) or a light absorbing protein(s).

Still an even further exemplary embodiment of the present disclosure canbe a label, which can include a chromophore(s) and an isotopicallymodified nitrile(s).

These and other objects, features and advantages of the exemplaryembodiments of the present disclosure will become apparent upon readingthe following detailed description of the exemplary embodiments of thepresent disclosure, as also exemplified by the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Further objects, features and advantages of the present disclosure willbecome apparent from the following detailed description taken inconjunction with the accompanying FIGS. showing illustrative exemplaryembodiments of the present disclosure, in which:

FIGS. 1 a-1 b show imaging complex protein metabolism by stimulatedRaman scattering (SRS) microscopy in live cells, tissues and animals.For example, FIG. 1 a illustrates a cartoon for SRS imaging followingmetabolic labeling of deuterated amino acids (D-AAs) in live organisms(e.g. mice), which are first administered with D-AAs for certain periodof time and then imaged by SRS to probe protein metabolism. FIG. 1 billustrates spontaneous Raman spectra from HeLa cells incubated withmedium containing either regular amino acids (gray, dashed) or D-AAsillustrate three distinct ways to probe complex protein metabolism:imaging newly synthesized proteins by targeting 2133 cm⁻¹ fromcarbon-deuterium bonds (C-D), imaging degradation of pre-existingproteins by targeting the pure methyl group (CH₃) distribution, andtwo-color pulse-chase protein imaging by labeling with two sub-groups ofD-AAs (i.e., group I and group II).

FIGS. 2 a-2 e depict sensitivity optimization and time-lapse imaging ofthe de novo proteome synthesis dynamics. For example, FIG. 2 aillustrates spontaneous Raman spectra of C-D peaks in HeLa cellsincubated in optimized deuteration medium display a 50% increase whencompared to the previously reported partial deuteration medium, andabout 8 times higher than using leucine-d₁₀ only. FIG. 2 b illustratesSRS images of newly synthesized proteins in live HeLa cells confirm a50% average signal increase. FIG. 2 c illustrates SRS images of newlysynthesized proteins in live neurons in optimized deuteration medium for20 h. The zoom-in image highlights the fine dendritic structures (likelydendritic spines, arrow-headed). FIG. 2 d illustrates SRS image of newlysynthesized proteins in live HeLa cells with 1 h incubation of optimizeddeuteration medium. Control image with protein synthesis inhibitiondeprives most of the signal. FIG. 2 e illustrates time-lapse SRS imagesof protein synthesis dynamics in a same set of live HeLa cells withcontinuous incubation in optimized deuteration medium. Scale bar, 10 μm.

FIGS. 3 a-3 c show time-dependent SRS images of protein degradation. Forexample, FIG. 3 a illustrates adopting a linear combination algorithmbetween 2940 cm⁻¹ and 2845 cm⁻¹ channels, the obtained SRS imageexclusively from CH₃ vibration display gradual degradation ofpre-existing proteins in live HeLa cells cultured in optimizeddeuteration medium for 0 h, 24 h, 48 h and 96 h. FIG. 3 b illustratesSRS images exclusively from CH₂ vibration display the total lipiddistribution at the corresponding time point. FIG. 3 c illustrates asingle exponential decay fitting from averaged image intensities frompre-existing protein in FIG. 3 a , yielding a protein degradation timeconstant of 45±4 h. Error bars, standard deviation. Scale bar, 10 μm.

FIGS. 4 a-4 d depict pulse-chase SRS imaging of temporally definedproteins. For example, FIG. 4 a illustrates structures and spontaneousRaman spectra of group I D-AAs (i.e. the branched chain amino acids).FIG. 4 b illustrates structures and spontaneous Raman spectra of threeexamples of group II non-branched D-AAs. FIG. 4 c illustratesspontaneous Raman spectra of HeLa cells cultured with group I D-AAs(element 405), showing multiple peaks with the first around 2067 cm⁻¹,and with group II D-AAs (element 410), showing a common peak around 2133cm⁻¹. FIG. 4 d illustrates two-color pulse-chase imaging by sequentiallabeling of group II and group I D-AAs in time with simultaneousexpression of mutant huntingtin (mHtt94Q-mEos2) proteins. Cartoondisplays experimental timeline of plasmid transfection and D-AA mediumexchanges. The fluorescence image (overlaid with bright field) indicatesthe formation of a large aggregate (arrow-headed) of mHtt94Q-mEos2. Theretrieved signals from linear combination of the original images at 2067and 2133 channels display a large aggregation of mHtt proteins solelylabeled by group II D-AAs during the first 22 h (pulse 415) and mHttonly labeled by group I D-AAs during the following 20 h (chase 420). Themerged image, as well as the intensity profile, from the pulsed (element415) and chased (element 420) images confirms with a yellow core and agreen shell. Scale bar, 10 μm.

FIGS. 5 a-5 b show SRS imaging of newly synthesized proteins in livemouse brain tissues. For example, FIG. 5 a illustrates SRS images atdentate gyrus of a live organotypic brain slice (400 μm thick, from aP10 mouse) after culturing in D-AA medium for 30 hr. 2133 cm⁻¹ (C-D)image presents the distribution of newly synthesized proteins. The CH₃and CH₂ images show the old protein pools and total lipids,respectively. FIG. 5 b illustrates a 4-by-3 mm large field view overlayimage of new proteins (C-D, element 505), old proteins (CH₃, element510) and total lipids (CH₂, element 515) for a brain slice (400 μmthick, from a P12 mouse) cultured in D-AA medium for 30 h. Scale bar,100 μm.

FIGS. 6 a-6 c show SRS imaging of newly synthesized proteins in vivo.For example, FIG. 6 a illustrates SRS images of a 24-hpf (hpf: hourspost fertilization) zebrafish. Wild-type zebrafish embryos were injectedat 1-cell stage with 1 nL D-AA solution and allowed to develop normallyfor another 24 h before imaging. Bright field image shows the grossmorphology of embryonic zebrafish at 24 hpf (dashed boxes). 2133 cm⁻¹(C-D) image presents the distribution of newly synthesized proteins(Supplemental FIG. 2 a ) in the somites of an embryonic zebrafish tail.The CH₃ image shows the old protein pool while the CH₂ image depictstotal lipid in the same fish. FIGS. 6 b and 6 c SRS images of live mouseliver are shown in FIG. 6 b and intestine tissues FIG. 6 c harvestedfrom mice after administered with D-AA containing drinking water for 12days. 2133 cm⁻¹ (C-D) channel shows newly synthesized proteins(Supplemental FIGS. 2 b-2 c that resemble the distribution of totalprotein as shown in the 1655 cm⁻¹ image (Amide I). Scale bar, 10 μm.

FIGS. 7 a-7 b depict SRS images at 2067 cm⁻¹ and 2133 cm⁻¹ channels ofproteins labeled with group I D-AA only shown in FIG. 7 a and group IID-AA only shown in FIG. 7 b.

FIGS. 8 a-8 c show raw C-D on-resonance (2133 cm⁻¹) and off-resonance(2000 cm⁻¹) SRS images of newly synthesized proteins in vivo in FIG. 6 a, which illustrates SRS C-D on-resonance and off-resonance images of a24 hpf embryonic zebrafish. The difference image between C-Don-resonance and off-resonance (pixel-by-pixel subtraction) shows pureC-D labeled protein distribution in the somites of an embryoniczebrafish tail, as in FIGS. 6 a-6 c . FIGS. 8 b and 8 c illustrate SRSC-D on-resonance and off-resonance images of live mouse liver FIG. 6 band intestine FIG. 6 c tissues harvested from the mice afteradministering with D-AA containing drinking water for 12 days. Thedifference image between C-D on-resonance and off-resonance(pixel-by-pixel subtraction) shows pure C-D labeled protein distributionin the liver and intestine tissues, shown in FIG. 6 b and FIG. 6 c ,respectively. The residual signal presented in the off-resonance imagesmainly comes from cross-phase modulation induced by highly scatteringtissue structures.

FIGS. 9 a-9 b show SRS imaging for newly synthesized proteins in vivowith intraperitoneal injection of mice with D-AA solutions. For example,FIGS. 9 a and 9 b illustrate SRS images of live mouse liver FIG. 9 a andintestine FIG. 9 b tissues harvested from mice after intraperitonealinjection injected with D-AAs solutions for 36 h. 2133 cm⁻¹ channelshows newly synthesized proteins (off-resonance image subtracted) thatresemble the distribution of total proteins as shown in the 1655 cm⁻¹image (Amide I). FIGS. 9 c and 9 d illustrate corresponding raw C-Don-resonance (2133 cm⁻¹) and off-resonance (2000 cm⁻¹) images are shownas references for liver FIG. 9 c and intestine FIG. 9 d tissues. Scalebar, 10 μm.

FIGS. 10 a-10 c depict bond-selective SRS imaging of alkynes asnonlinear vibrational tags. For example, FIG. 10 a illustratesSpontaneous Raman spectra of HeLa cells and 10 mM EdU solution. Inset:the calculated SRS excitation profile (FWHM 6 cm⁻¹) is well fittedwithin the 2125 cm⁻¹ alkyne peak (FWHM 14 cm⁻¹, magenta). FIG. 10 billustrates linear dependence of stimulated Raman loss signals (2125cm⁻¹) with EdU concentrations under a 100 μs acquisition time. FIG. 10 cillustrates the metabolic incorporation scheme for a broad spectrum ofalkyne-tagged small precursors. a.u. arbitrary units.

FIG. 11 illustrates the working mechanism of a stimulated Ramanscattering with A Pump beam (pulsed, pico-second) and anintensity-modulated Stokes beam (pulsed, pico-second). The Pump beam(pulsed, pico-second) and an intensity-modulated Stokes beam (pulsed,pico-second) are both temporally and spatially synchronized beforefocused onto cells that have been metabolically labeled withalkyne-tagged small molecules of interest. When the energy differencebetween the Pump photon and the Stokes photon matches the vibrationalfrequency (Ω_(ab)) of alkyne bonds, alkyne bonds are efficiently drivenfrom their vibrational ground state to their vibrational excited state,passing through a virtual state. For each excited alkyne bond, a photonin the Pump beam is annihilated (Raman loss) and a photon in the Stokesbeam is created (Raman gain). The detected pump laser intensity changesthrough a lock-in amplifier targeted at the same frequency as themodulation of Stokes beam serve as the contrast for alkynedistributions.

FIGS. 12 a-12 j show live SRS imaging of de novo synthesis of DNA, RNA,proteomes, phospholipids and triglycerides by metabolic incorporation ofalkyne-tagged small precursors. For example, FIG. 12 a illustrates Ramanspectra of cells incubated with EdU, EU, Hpg, propargylcholine and17-octadecynoic acid (17-ODYA). FIG. 12 b illustrates live HeLa cellsincubated with 100 μM EdU alone (alkyne-on) and with 10 mM hydroxyurea(Control). FIG. 12 c illustrates time-lapse images of a dividing cellincubated with 100 μM EdU. FIG. 12 d illustrates live HeLa cellsincubated with 2 mM EU alone (alkyne-on) and with 200 nM actinomycin D(Control). FIG. 12 e illustrates pulse-chase imaging of RNA turnover inHeLa cells incubated with 2 mM EU for 12 h followed by EU-free medium.FIG. 12 f illustrates live HeLa cells incubated with 2 mM Hpg alone(alkyne-on) and with 2 mM methionine (Control). FIG. 12 g illustrateslive neurons incubated with 1 mM propargylcholine (alkyne-on). FIG. 12 hillustrates live macrophages incubated with 400 μM 17-ODYA (alkyne-on).FIG. 12 i , illustrates C. elegans fed with 17-ODYA (alkyne-on). FIG. 12j illustrates dual-color images of simultaneous EdU (2125 cm⁻¹) andpropargylcholine (2142 cm⁻¹) incorporation. For FIG. 12 b , FIG. 12 d ,and FIG. 12 f , alkyne-off and amide images display the same set ofcells as the alkyne-on images; lipid images capture the same cells ascontrol images. Scale bars, 10 μm. Representative images of 10-15trials. a.u.=arbitrary units.

FIG. 13 shows SRS imaging of distal mitotic region of C. elegansgermline incorporated with EdU. The composite image shows both theprotein derived 1655 cm⁻¹ (amide) signal from all the germ cells, andthe direct visualization of alkynes (2125 cm⁻¹ (EdU)) highlighting theproliferating germ cells. White circles show examples of EdU positivegerm cells in the mitotic region of C. elegans germline. Scale bar, 5μm.

FIG. 14 shows SRS imaging of fixed HeLa cells after incorporating with 2mM Hpg. The alkyne-on image displays the Hpg distribution for the newlysynthesized proteins. For the same set of cells, the off-resonant(alkyne-off) image shows vanishing signal, and the amide image showstotal protein distribution. This result confirms that the detectedsignal is not from freely diffusive precursor Hpg itself (which iseliminated during the fixation process). Scale bar, 10 μm.

FIGS. 15 a-15 c show click-chemistry based fluorescence staining offixed HeLa cells. Fluorescence images of HeLa cells incorporated withFIG. 15 a , EdU (for DNA); FIG. 15 b EU (for RNA); FIG. 15 c Hpg (forprotein). Scale bars, 10 μm.

FIGS. 16 a-16 c show SRS imaging of propargylcholine incorporation inNIH3T3 cells and control experiments. For example, FIG. 16 a illustratesfixed NIH3T3 cells after culturing with 0.5 mM propargylcholine for 48hours. The alkyne-on image shows alkyne-tagged choline distribution.FIG. 16 b illustrates treatment of fixed NIH3T3 cells with phospholipaseC, which removes Choline head groups of phospholipids only in thepresence of calcium. The alkyne-on image shows the strong decrease ofincorporated propargylcholine signal, supporting its main incorporationinto membrane phospholipids. FIG. 16 c illustrates treatment of fixedNIH3T3 cells with phospholipase C in the presence of EDTA (chelatingcalcium). Propargylcholine signal is retained in the alkyne-on image.FIGS. 16 a-16 c illustrate images in the same set of cells as inalkyne-on images, the alkyne-off images show a clear background. Theamide images display total protein distribution. Scale bars, 10 μm.

FIGS. 17 a-17 e show in vivo delivery of an alkyne-bearing drug (TH inDMSO) into mouse ear. For example, FIG. 17 a illustrates Raman spectraof a drug cream, Lamisil, containing 1% TH and mouse ear skin tissue.FIGS. 17 b-17 e illustrate SRS imaging of tissue layers from stratumcorneum (z=4 μm) to viable epidermis (z=24 μm), sebaceous gland (z=48μm) and subcutaneous fat (z=88 μm). To facilitate tissue penetration,DMSO solution containing 1% TH was applied onto the ears of ananesthetized live mouse for 30 min and the dissected ears are imagedafterwards. For all 4 layers: alkyne-on images display TH penetration;alkyne-off images show off-resonant background (The bright spots in FIG.18 b are due to two-photon absorption of red blood cells). The compositeimages show protein (1655 cm⁻¹) and lipid (2845 cm⁻¹) distributions.Scale bars, 20 μm. a.u. arbitrary units.

FIGS. 18 a-18 b show in vivo delivery of an alkyne-bearing drug (TH inLamisil cream, a FDA approved drug cream) into mouse ear. For example,FIG. 18 (a-b) illustrates SRS imaging of the viable epidermis layer(z=20 μm) and the sebaceous gland layer (z=40 μm). For both FIG. 18 aand FIG. 18 b : illustrates the alkyne-on images display the THpenetration into mouse ear tissues through lipid phase. The compositeimages show both protein (1655 cm⁻¹) and lipid (2845 cm⁻¹)distributions. Scale bars, 20 μm.

FIG. 19 shows an exemplary synthesis route for a bond-edited compound.

FIGS. 20 a-20 b show in FIG. 20 a another exemplary synthesis route fora bond-edited compound (alkyne-D-glucose) and in FIG. 20 b thespectroscopic characterization of the bond-edited compound in PBS bufferand in mammalian cells.

FIG. 21 shows time-dependent alkyne-D-glucose (32 mM) uptake in liveHeLa cells at 10 min, 30 min, 1 h, 2 h, 3 h and 4 h time points. Theglucose signal inside mammalian cells is increasing over time.

FIG. 22 shows the results of a competition experiment to confirm theuptake of alkyne-D-glucose. Regular D-glucose is added into cell mediumfor HeLa cells to compete with the uptake of alkyne-D-glucose. With theincreasing concentration of regular D-glucose (10 mM, 50 mM and 100 mM),the alkyne-D-glucose signal decreases (as shown both in images and bardiagrams). When using L-glucose (which cells do not uptake) ascompetition for alkyne-glucose, the alkyne-D-glucose signal is retained.

FIGS. 23 a-23 b show alkyne-glucose uptake in both neuronal culture FIG.23 a and brain slices FIG. 23 b.

FIG. 24 shows multicolor imaging of DNA synthesis with EdU (1), EdU-¹³C(2) and EdU-¹³C2 (3).

FIG. 25 shows pulse-chase imaging of DNA synthesis (EdU (1) for pulseand EdU-¹³C2 (3) for chase). The merged images show that the twocompounds can label two temporally different cells populations for DNAsynthesis.

FIG. 26 shows simultaneous three-color chemical imaging using alkyneprobes for DNA synthesis (EdU-¹³C (2) at 2077 cm-1) and RNA synthesis(EU-¹³C2 (13) at 2053 cm-1 and 17-ODYA (12) at 2125 cm-1).

FIG. 27 shows images of subcutaneous colon cancer. Subcutaneous coloncancer was grown for 15 days in mice, dissected out and cultured ex vivoin deuterated amino acids containing medium for 47 h (400 um thick).Live image of the tumors shows intensive protein synthesis activity.

FIGS. 28 a-28 b show active glucose metabolism in HeLa cells cultured indeuterated glucose medium. For example, FIG. 28 a illustrates imagesafter culturing in 0.1% D7-Glucose in EMEM for 48 hrs. FIG. 28 billustrates HeLa cell images after culturing 0.2% D7-Glucose in EMEM for48 hrs.

FIG. 29 shows active glucose metabolism in tumor cell line U87MGcultured in deuterated glucose medium (0.1% D7-Glucose in EMEM) for 48hrs.

FIG. 30 shows the detection of D2O as a labeling reagent of themetabolism with stimulated Raman scattering.

FIG. 31 shows SRS imaging of C-D formation using D2O as a metabolicreagent for various of live organisms.

FIGS. 32 a-32 c show imaging of 13C-phenylalanine labeled proteins forprotein turnover. For example, FIG. 32 a shows an illustration of aspectroscopic characterization of Raman shift from 1004 cm-1 to 968 cm⁻¹with the labeling of ¹³C-phenylalanine, and FIGS. 32 b and 32 cillustrate a time dependent ¹³C-phenylalanine labeling, whereas FIG. 32b shows a spectrum and FIG. 32 c illustrates SRS images, where the 968cm-1 signal for ¹³C labeled proteins are increasing while the 1004 cm⁻¹signal of old ¹²C-proteins are decreasing.

FIGS. 33 a-f are a set of exemplary images based on SRS imaging of newlysynthesized proteins by metabolic incorporation of deuterium-labeled allamino acids in live HeLa cells. For example, FIG. 33 a illustratesSpontaneous Raman spectrum of HeLa cells incubated with a mediumcontaining deuterium-labeled all amino acids for 20 hrs, showing a ˜5times stronger peak at 2133 cm⁻¹ than the spectrum in FIG. 2 a . FIG. 33b illustrates SRS image targeting the central 2133 cm⁻¹ vibrational peakof C-D shows a high-contrast image representing newly synthesizedproteins. The same intensity scale bar is used here as in FIG. 2 a .Consistent with previous reports, nascent proteins are distributed witha higher percentage in nucleoli (indicated by arrows) which are theactive sites for ribosome biogenesis involving rapid import anddegradation of proteins. FIG. 33 c illustrates SRS image of the samecells as in FIG. 33 b at off-resonance frequency 2000 cm⁻¹ isbackground-free. FIGS. 33 d-33 f illustrate SRS images of same cells asin FIG. 33 b at frequency 1655 cm⁻¹ (amide I stretching attributedprimarily to proteins); 2845 cm⁻¹ (CH₂ stretching attributed mainly tolipids) and 2940 cm⁻¹ (CH₃ stretching attributed mainly to proteins)show the intrinsic distributions of total cellular lipids and proteins.

FIGS. 34 a-34 m are a set of exemplary images based on SRS imaging oftime-dependent de novo protein synthesis and drug-induced proteinsynthesis inhibition effect in live HeLa cells incubated indeuterium-labeled all amino acid medium. For example, FIGS. 34 a-34 fSRS image targeting the central 2133 cm⁻¹ vibrational peak of C-Ddisplays a time-dependent signal increase (5 hrs—FIG. 34 a, 12 hrs—FIG.34 b, 20 hrs—FIG. 34 c ) of the newly synthesized proteins, withnucleoli being gradually highlighted. As a control, the amide I (1655cm⁻¹) signal remains at a steady state over time (5 hrs—FIG. 34 d, 12hrs—FIG. 34 e, 20 hrs—FIG. 340 . FIGS. 34 g-34 i illustrate ratio imagesbetween the SRS image at 2133 cm⁻¹ (newly synthesized proteins) and theSRS image at 1655 cm⁻¹ (the amide I band from total proteins),representing the relative new protein fraction with subcellularresolution at each time point (5 hrs—FIG. 34 g, 12 hrs—FIG. 34 h, 20hrs—FIG. 34 i ). The bar represents the ratio ranging from low to high.FIG. 34 j shows time-lapse SRS images of a live dividing HeLa cellduring a 25 min time-course after 20-hour incubation with deuterated allamino acids medium. FIG. 34 k illustrates a spontaneous Raman spectrumof HeLa cells incubated with both deuterium-labeled all amino acids anda protein synthesis inhibitor anisomycin (5 μM) for 12 hrs shows thedrastic attenuation of the C-D Raman peak at 2133 cm⁻¹. FIG. 34 l showsan exemplary SRS image of the same sample displays near vanishing signalthroughout the whole field of view. FIG. 34 m shows, as a control, theimage of the same cells at 2940 cm⁻¹ confirms that anisomycin does notinfluence the total protein level.

FIGS. 35 a-35 f are a set of exemplary images based on SRS imaging ofnewly synthesized proteins by metabolic incorporation ofdeuterium-labeled all amino acids in live human embryonic kidney(HEK293T) cells. For example, FIG. 35 a illustrates the spontaneousRaman spectrum of HEK293T cells incubated with deuterium-labeled allamino acids for 12 hrs shows a 2133 cm⁻¹ C-D peak nearly as high as theAmide I (1655 cm-1) peak. FIG. 35 b shows an exemplary SRS imagetargeting the central 2133 cm⁻¹ vibrational peak of C-D shows newlysynthesized proteins in live HEK293T cells displaying a similar signallevel as HeLa cells at 12 hrs (FIG. 4 b ). FIG. 35 c shows, as acomparison, the off-resonant image is still background-free. FIGS. 35 dand 35 e illustrate multicolor SRS images of intrinsic cell molecules:total proteins (1655 cm⁻¹ (FIG. 35 d ) and lipids (2845 cm⁻¹ (FIG. 35 e). FIG. 35 f illustrates the ratio image between new proteins (2133cm-1) and total proteins (1655 cm-1) illustrates a spatial map fornascent protein distribution.

FIGS. 36 a-36 f are a set of exemplary images based on SRS imaging ofnewly synthesized proteins in both cell bodies and newly grown neuritesof neuron-like differentiable mouse neuroblastoma (N2A) cells. Duringthe cell differentiation process by serum-deprivation and 1 μM retinoicacid, deuterium-labeled all amino acids medium is also supplied for 24hrs. For example, FIG. 36 a illustrates SRS images targeting the 2133cm⁻¹ peak of C-D show newly synthesized proteins. FIG. 36 b illustratesSRS images targeting the 2940 cm⁻¹ CH₃ show total proteins. FIGS. 36 cand 36 d illustrate zoomed-in images as indicated in the white dashedsquares in FIGS. 36 a and 36 b . FIG. 36 e illustrates a ratio imagebetween new protein FIG. 36 c and total proteins FIG. 36 d . While thestarred neurites show high percentage of new proteins, the arrowsindicate neurites displaying very low new protein percentage. FIG. 36 fmerged image between new protein c (channel 3605) and total proteins inFIG. 36 d (channel 3610). Similarly, starred regions show obvious newproteins; while arrows indicate regions that have undetectable newprotein signal.

FIG. 37 a is a prior art recipe for a mammalian cell culture.

FIG. 37 b is an exemplary deuterium-labeled recipe based on the cellculture of FIG. 36 ;

FIG. 38 is an illustration of an exemplary block diagram of an exemplarysystem in accordance with certain exemplary embodiments of the presentdisclosure;

FIG. 39 a is diagram of an exemplary SRS system according to anexemplary embodiment of the present disclosure;

FIGS. 39 b-39 e are exemplary illustrations of energy outputs accordingto an exemplary embodiment of the present disclosure;

FIGS. 40 a-40 d are exemplary graphs of signal sensitivities accordingto an exemplary embodiment of the present disclosure;

FIGS. 41 a-41 j are images taken with the exemplary SRS system accordingto an exemplary embodiment of the present disclosure;

FIG. 42 a is a chart of pr-SRS spectra according to an exemplaryembodiment of the present disclosure;

FIGS. 42 b-42 d are images taken using the exemplary pr-SRS systemaccording to an exemplary embodiment of the present disclosure;

FIGS. 43 a-43 c are diagrams of exemplary dye molecules according to anexemplary embodiment of the present disclosure;

FIG. 43 d is an exemplary graph of the wavenumbers versus normalizedintensity of the exemplary dye molecules from FIGS. 43 a-43 c accordingto an exemplary embodiment of the present disclosure; and

FIG. 44 is a set of further images taken using the exemplary pr-SRSsystem according to an exemplary embodiment of the present disclosure.

Throughout the drawings, the same reference numerals and characters,unless otherwise stated, are used to denote like features, elements,components or portions of the illustrated exemplary embodiments.Moreover, while the present disclosure will now be described in detailwith reference to the figures it is done so in connection with theillustrative exemplary embodiments and is not limited by the particularexemplary embodiments illustrated in the figures, and provided in theappended claims.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

The following detailed description is presented to enable any personskilled in the art to make and use the present disclosure. For purposesof explanation, specific nomenclature is set forth to provide a thoroughunderstanding of the present invention. However, it will be apparent toone skilled in the art that these specific details are not required topractice the invention. Descriptions of specific applications areprovided only as representative examples. The present disclosure is notintended to be limited to the exemplary embodiments shown, but is to beaccorded the widest possible scope consistent with the principles andfeatures disclosed herein.

As used herein, the term “Raman scattering” refers to a spectroscopictechnique used to observe vibrational, rotational, and otherlow-frequency modes in a system. It relies on inelastic scattering, orRaman scattering, of monochromatic light, usually from a laser in thevisible, near infrared, or near ultraviolet range. The laser lightinteracts with molecular vibrations, phonons or other excitations in thesystem, resulting in the energy of the laser photons being shifted up ordown. The shift in energy gives information about the vibrational modesin the system. A variety of optical processes, both linear and nonlinearin light intensity dependence, are fundamentally related to Ramanscattering. As used herein, the term “Raman scattering” includes, but isnot limited to, “stimulated Raman scattering” (SRS), “spontaneous Ramanscattering”, “coherent anti-Stokes Raman scattering” (CARS),“surface-enhanced Raman scattering” (SERS), “Tip-enhanced Ramanscattering” (TERS) or “vibrational photoacoustic tomography”.

The exemplary system, method and computer accessible medium, accordingto an exemplary embodiment of the present disclosure, can use alkyne asa vibrational tag coupled with narrow-band stimulated Raman scatteringmicroscopy (“SRS”) for the detection of small molecules insidebiological systems. The use of alkyne as a vibrational tag (e.g. a Ramantag) offers a large Raman cross-section enabling sensitive detection(See, e.g., References 13; 14). Additionally, the alkyne Raman peak canexhibit a narrow spectral width for the specific detection, which canreduce the probability of overlapping with other tags. Furthermore, theRaman peak of alkyne can lay exactly in the cell-silent region in thecell spontaneous Raman spectrum, bypassing the complex interference fromvast pool of biomolecules in the fingerprint region. (See, e.g., FIG.1B).

The exemplary SRS can be a sensitive vibrational imaging microscopy. Byharnessing Einstein's stimulated emission process, the exemplary SRS canemploy two-laser excitation (e.g., temporally and spatially overlappedPump and Stokes lasers), boosting up the transition rate about 7 ordersof magnitude as compared to the traditional spontaneous Ramanmicroscopy, the transition process of which can be intrinsically weak(e.g., 10 to 12 orders of magnitude slower than fluorescence). (See,e.g., References 6; 8; 15). The exemplary SRS can be a bond-selectiveprocedure with high specificity, in contrast with the spontaneous Ramanimaging which can be a spectrum-based method. Instead of spreading theenergy to the whole spectrum as in the spontaneous Raman imaging, theexemplary narrow-band SRS can focus its energy to the vibrationaltransition of a specific bond. A 6-ps pulse width can be chosen for bothSRS pump and stokes lasers to achieve a spectral resolution of 5 cm⁻¹for the detection of alkyne. The spectral width of the excitationprofile from two combined lasers can be calculated to be 8 cm⁻¹, whichcan fit well within the spectral width of alkyne Raman peak that can be14 cm⁻¹. (See, e.g., FIG. 1B). Hence, the exemplary laser pulse widthcan be long enough that all the laser energy can be used to specificallydetect alkyne without energy waste, but short enough that the two-photonefficiency can be maintained since the exemplary SRS can depend on anonlinear process.

The exemplary SRS signal can offer linear concentration dependence tothe analyte without non-specific background. Compared to a previouslyknown nonlinear vibrational imaging procedure such coherent anti-StokesRaman scattering (“CARS”) microscopy, which suffers from spectraldistortion, unwanted non-resonant background, non-straightforwardconcentration dependence and coherent image artifact, the exemplary SRScan exhibit straightforward image interpretation and quantificationwithout complications from non-resonant background and phase-matchingconditions (See, e.g., References 7; 8; 16). Besides the above-mentionedadvantages, SRS can also have its own distinctive characters as animaging procedure. For example, SRS can be immune to fluorescencebackground as compared to spontaneous Raman microscopy that can sufferfrom large fluorescence background. In addition, SRS, as a nonlinearprocess, can offer intrinsic 3D sectioning capability. Moreover, byadopting near-infrared excitation, SRS can offer deeper penetrationdepth and less photo-toxicity, which can be well suited for imaging livecells, tissues and animals. Recently, narrow-band SRS has achievedunprecedented sensitivity down to approximately 1000 retinoic acidmolecules and up to video rate imaging speed in vivo. (See, e.g.,Reference 17).

Alkyne can be a metabolic labeling tag in fluorescence microscopyutilizing click-chemistry with azide-linked fluorescent tags (See, e.g.,References 18-23). Unfortunately, this type of click-chemistry basedfluorescence detection usually requires non-physiological fixation andsubsequent dye staining and washing. The exemplary Raman detection, incontrast, does not have such requirements, since it can directly imagevibrational modes of alkyne, bypassing the subsequent additionalprocesses.

All of the above applications can show the universal and distinctadvantage of the exemplary SRS coupled with alkyne tags to image thesmall molecule metabolites dynamics and drug distributions in the livecells, organisms and animals with minimum perturbation and highspecificity and sensitivity, extending the repertoire of reporters forbiological imaging beyond fluorophores.

Method for Obtaining Biological Information in a Living Cell or a LivingOrganism with Bond-Edited Compounds

One aspect of the present disclosure relates to a method for obtainingbiological information in a living cell or a living organism withbond-edited compounds using Raman scattering. The method comprises thesteps of introducing an effective amount of one or more bond-editedcompounds into a live cell or a living organism, and detecting avibrational tag in the cell or organism with Raman scattering. In someexemplary embodiments, the Raman scattering is SRS.

The term “biological information” as used herein, refers to spatialdistribution of the targeted molecules, such as one-dimensional line, ortwo-dimensional or three-dimensional images, and non-imaginginformation, such as a simple signal intensity or local spectrum on asingle location or its time dependence.

As used herein, the term “bond-edited compounds” refers to compoundshaving one or more chemical bond that may serve as a vibrational tag fordetection by Raman scattering. Examples of chemical bond that may serveas a vibrational tag include, but are not limited to, carbon-carbontriple bond, carbon-nitrogen triple bond, azide bond, carbon-deuteriumbond, phenol ring, ¹³C modified carbon-carbon triple bond, ¹³C modifiedcarbon-nitrogen triple bond, ¹³C modified azide bond, ¹³C modifiedcarbon-deuterium bond, ¹³C modified phenol ring and combinationsthereof.

As used herein, the term “effective amount” refers to an amount that,when introduced into a live cell or organism, is sufficient to reach aworking concentration needed for SRS imaging. The “effective amount”would vary based on the type of bond-edited compound, as well as thecells or organisms that the bond-edited compound is introduced into. Insome embodiments, an “effective amount” of a bond-edited compound is theamount that is sufficient to reach an in vivo concentration of 1 μM to100 mM, 3 μM to 30 mM, 10 μM to 10 mM, 100 μM to 1 mM, 10 μM to 1 mM or10 μM to 100 μM in a target cell or organ. In some embodiments, an“effective amount” of a bond-edited compound comprising a triple bond isthe amount that is sufficient to reach an in vivo concentration of 1 μMto 10 mM, 3 μM to 3 mM, 1 μM to 1 mM or 30 μM to 300 μM. In someembodiments, an “effective amount” of a bond-edited compound comprisinga triple bond is the amount that is sufficient to reach an in vivoconcentration of about 100 μM. In other embodiments, an “effectiveamount” of a bond-edited compound comprising a C-D bond is the amountthat is sufficient to reach an in vivo concentration of 10 μM to 100 mM,30 μM to 30 mM, 100 μM to 10 mM or 300 μM to 3 mM. In some embodiments,an “effective amount” of a bond-edited compound comprising a C-D bond isthe amount that is sufficient to reach an in vivo concentration of about1 mM.

In some exemplary embodiments, the bond-edited compounds are smallmolecules. As used herein, the term “small molecules” refers to lowmolecular weight organic compound having a molecular weight of 1000daltons or less. In some exemplary embodiments, the small molecules havea size on the order of 10⁻⁹ m. Examples of small molecules include, butare not limited to, water, ribonucleosides, ribonucleotides,deoxyribonucleoside, deoxyribonucleotide, amino acids, peptides,choline, monosaccharides, disaccharides, fatty acids, glucose, adenosinetriphosphate, adenosine diphosphate, cholesterol, neurotransmitters,secondary messengers, and chemical drugs.

In some exemplary embodiments, said bond-edited compound contains one,two, three, four, five, six, seven, eight, nine, ten or more vibrationaltags. The vibrational tags may be the same type of tags or a mixture ofone or more different tags.

In some exemplary embodiments, said vibrational tag is an alkyne tag. Inother exemplary embodiments, said vibrational tag is an azide tag. Instill other exemplary embodiments, said vibrational tag is an isotopelabel. In a further exemplary embodiment, said isotope label is acarbon-deuterium tag. In yet still other exemplary embodiments, saidvibrational tag is a combination of an alkyne tag and a carbon-deuteriumtag.

In particular exemplary embodiments, said at least one vibrational tagcomprises at least one vibrational tag selected from the groupconsisting of —C≡C—, —C≡N, —N═N═N, —C≡C—C≡C—, —C≡C—C≡N, —C-D, and—C≡C-D.

In a further exemplary embodiment, the vibrational comprises at leastone ¹³C atom or one deuterium atom.

In some exemplary embodiments, the bond-edited compound is an aminoacid.

In further exemplary embodiments, the amino acid is an essential aminoacid.

In a still further exemplary embodiment, the essential amino acid isselected from the group consisting of histidine, isoleucine, leucine,lysing, methionine, phenylalanine, threonine, tryptophan and valine.

In other exemplary embodiments, the bond-edited compound is a nucleosideor a nucleotide.

In still other exemplary embodiments, the bond-edited compound is afatty acid.

In still other exemplary embodiments, the bond-edited compound is amonosaccharide or a disaccharide. In a further exemplary embodiment, thebond-edited compound is glucose, a glucose derivative or propargylglucose.

In still other exemplary embodiments, the bond-edited compound is apharmaceutical agent, such as an anti-cancer agent, anti-inflammatoryagent, anti-bacterial agent, anti-fungal agent and anti-viral agent.

In still other exemplary embodiments, the bond-edited compound is acytokine or chemokine.

In some exemplary embodiments, the bond-edited compound is EU-¹³C₂having a molecular structure of formula 13:

In some exemplary embodiments, the bond-edited compound is EdU-¹³C₂having a molecular structure of formula 3:

In some exemplary embodiments, the bond-edited compound is EdU-¹³Chaving a molecular structure of formula 2:

In some exemplary embodiments, the bond-edited compound is EdU-¹³C′having a molecular structure of formula 14:

In some exemplary embodiments, the bond-edited compound isalkyne-D-glucose having a molecular structure of formula S3:

In some exemplary embodiments, the bond-edited compound is metabolizedin the living cell or organism and the vibrational tag is transferredfrom the bond-edited compound to a down-stream metabolite of thebond-edited compound (See, e.g., FIGS. 28-31 ).

In still other exemplary embodiments, the method comprises introducinginto a live cell a mixture of bond-edited compounds that imaging withRaman scattering at two or more different wavelengths. In some relatedexemplary embodiments, the Raman scattering is SRS.

In still other exemplary embodiments, the method comprises introducinginto a live cell a mixture of different bond-edited compounds that allowmultiple color imaging with Raman scattering. In some related exemplaryembodiments, the Raman scattering is SRS. In a particular exemplaryembodiment, the mixture of different bond-edited compounds comprisesEU-¹³C₂, EdU-¹³C and 17-ODYA.

In some exemplary embodiments, the two or more bond-edited compoundstarget the same cellular component but at different time period (See,e.g., FIG. 25 ).

In still other exemplary embodiments, the method comprises introducinginto a living cell a mixture of different bond-edited compounds thattarget different cellular components.

In still other exemplary embodiments, the method comprises introducinginto a living organism a mixture of different bond-edited compounds thattarget different types of cells in the living organism.

In still other exemplary embodiments, the method comprises introducinginto a living organism a mixture of different bond-edited compoundscarrying different vibrational tags, and detecting the differentvibrational tags with Raman scattering using a linear combinationalgorithm. In some related exemplary embodiments, the Raman scatteringis SRS.

Method for Making Bond-Edited Compounds

Another exemplary aspect of the present disclosure relates to a methodfor making a bond-edited compound.

In one exemplary embodiment, the bond-edited compound is synthesized bythe route illustrated in FIG. 19 . In another exemplary embodiment, thebond-edited compound is synthesized by the route illustrated in FIG. 20a.

Method of Detecting Disease Conditions

Another exemplary aspect of the present disclosure relates to a methodfor detecting a disease condition in a subject, comprising:administering to said subject a composition comprising a bond-editedcompound targeting a disease tissue or pathogen, and detecting saidbond-edited compound by Raman scattering.

In some exemplary embodiments, the subject is a mammal. Exemplary mammalsubjects for use in accordance with the methods described herein includehumans, monkeys, gorillas, baboons, zoo animals and domesticatedanimals, such as cows, pigs, horses, rabbits, dogs, cats, goats and thelike.

In some exemplary embodiments, the disease condition is cancer.

In some exemplary embodiments, the disease condition is aneurodegenerative disease. In further exemplary embodiments, theneurodegenerative disease is selected from the group consisting ofamyotrophic lateral sclerosis, Parkinson's, Alzheimer's andHuntington's.

In some exemplary embodiments, the disease condition is an inflammatorydisease.

In some exemplary embodiments, the disease condition is a microbialinfection.

In some exemplary embodiments, the disease condition is a bacterialinfection.

In some exemplary embodiments, the disease condition is a viralinfection.

In some exemplary embodiments, the disease condition is a fungalinfection.

In some exemplary embodiments, the pathogen comprises bacteria.

Method for Monitoring Treatment for a Disease Condition

Another exemplary aspect of the present disclosure relates to a methodfor monitoring treatment for a disease condition. The method comprisesadministering to said subject a composition comprising a bond-editedcompound and detecting said bond-edited compound by SRS at a first timepoint, further administering to said subject said composition comprisinga bond-edited compound and detecting said bond-edited compound by Ramanscattering at a second time point, and comparing images obtained at thetwo time points.

In some exemplary embodiments, the first time point is a time point thatis about or prior to the initiation of a treatment and the second timepoint is a time point that is after the initiation of the treatment.

In other exemplary embodiments, the first time point and the second timepoint are two time points during the course of a treatment.

In some exemplary embodiments, the treatment is a treatment for cancer.

In other exemplary embodiments, the treatment is a treatment for aninflammatory disease.

In other exemplary embodiments, the treatment is a treatment for aneurodegenerative disease.

Method for Screening an Agent

Another exemplary aspect of the present disclosure relates to a methodfor screening an agent. The method comprises administering said agentand at least one bond-edited compound to a live cell or organism,detecting the bond-edited compound in the live cell or organism usingRaman scattering, and selecting a candidate agent based on one or morepredetermined criteria, such as the uptake, accumulation, trafficking,or degradation of the said bond-edited compound in the said live cell ororganism.

In some exemplary embodiments, the candidate agent is an anti-cancerdrug.

In some exemplary embodiments, the bond-edited compound is selected fromthe group consisting of amino acid, nucleic acid, ribonucleic acid andglucose derivatives.

In some exemplary embodiments, the candidate agent is a skinregenerating agent.

In some exemplary embodiments, the candidate agent is a cosmetic agent.

Method for Tracing a Cellular Process in a Live Cell with RamanScattering

Another exemplary aspect of the present disclosure relates to a methodfor tracing a cellular process in a live cell with Raman scattering. Themethod comprises introducing into a live cell a bond-edited compound,and following the physical movement or the chemical reaction or thebiological interaction of the bond-edited compound within the cell bySRS.

In some exemplary embodiments, the cellular processes are selected fromthe group consisting of DNA replication, RNA synthesis, proteinsynthesis, protein degradation, glucose uptake and drug uptake.

Composition for Labeling Cells with Bond-Edited Compounds

Another exemplary aspect of the present disclosure relates to acomposition for labeling a target cell with at least one bond-editedcompound. In some exemplary embodiments, the composition is a culturemedium comprising at least one bond-edited compound containing at leastone vibrational tag. The at least one bond-edited compound may beselected based on the type of the target cell or a target component(s)within the target cell.

In some exemplary embodiments, the culture medium comprises a pluralityof amino acids, wherein over 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% ofthe amino acids are tagged with one or more vibrational tag. In otherexemplary embodiments, the culture medium comprises a plurality of aminoacids, wherein all amino acids are tagged with one or more vibrationaltag.

In some exemplary embodiments, the culture medium comprises two, three,four, five, six, seven, eight, nine, ten or more different bond-editedcompounds.

Device for Imaging a Living Cell or a Living Organism with Bond-EditedCompounds

Another exemplary aspect of the present disclosure relates to a devicefor imaging bond-edited compounds by Raman scattering. The devicecomprises a first single-wavelength laser source that produces a pulselaser beam of a first wavelength, a second single-wavelength lasersource that produces a pulse laser beam of a second wavelength, amodulator that modulates either the intensity or the frequency or thephase or the polarization or the combination of the above of the pulselaser beam of one of the first or second laser source, a photodetectorthat is capable of detecting SRS or CARS or spontaneous Raman scatteringor the combination of the above from a biosample, and a computer.

In some exemplary embodiments, the energy difference between the photonsproduced by the first laser radiation and the photon produced by thesecond laser radiation matches with the energy of the vibrationaltransitions of the targeted vibrational tags. Photodetector of SRSdetects part or all of the first laser beam or the second laser beam.The output of the photodetector (which could be a photodiode) is furtherprocessed by a lock-in amplifier or a resonant circuit.

Another exemplary aspect of the present disclosure relates to anapparatus for providing radiation to at least one structure, comprising:a radiation providing arrangement which is configured to provide a pumpradiation and a stokes radiation, each at a fixed wavelength, whoseenergy difference is between about 2000 and 2500 wavenumbers.

In some exemplary embodiments, the radiation providing arrangement is alaser source.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tocarry out the method of the present disclosure and is not intended tolimit the scope of the invention. Efforts have been made to ensureaccuracy with respect to numbers used (e.g., amounts, temperature,etc.), but some experimental error and deviation should be accountedfor. Unless indicated otherwise, parts are parts by weight, molecularweight is weight average molecular weight, temperature is in degreesCentigrade and pressure is at or near atmospheric.

EXAMPLES Example 1: In Vitro and In Vivo Labeling with Deuterium Tags

In the examples described below, three major technical advances arebeing implemented, together with a series of biological applications oncomplex tissues and model animals in vivo (FIG. 1 a ). First, weoptimized the chemical composition of the deuterated culture medium thatachieved much higher deuterium labeling efficiency, and improved imagingsensitivity and speed of our SRS instrumentation. These optimizationsallow us to demonstrate time-lapse imaging of protein synthesis dynamicswithin single live cells. Second, we successfully imaged proteindegradation in live HeLa cells by targeting Raman peak of methyl group(CH₃) for the pre-existing protein pools and then employing a recentlydeveloped linear combination algorithm on measured SRS images at 2940cm⁻¹ and 2845 cm⁻¹ channels. Third, inspired by the classic pulse-chaseanalysis of complex protein dynamics, two-color pulse-chase imaging wasaccomplished by rationally dividing D-AAs into two structurallydifferent sub-sets that exhibit resolvable vibrational modes, asdemonstrated by tracking aggregate formation of mutant huntingtin (mHtt)proteins. Finally, going beyond the cellular level to visualizing morecomplex tissues and animals in vivo, we imaged the spatial distributionof newly synthesized proteins inside live brain tissue slices and inboth developmental embryonic zebrafish and mice (FIG. 1B). Takentogether, these technical advances and biological applicationsdemonstrate SRS microscopy coupled with metabolic labeling of D-AAs as acomprehensive and generally applicable imaging platform to evaluatecomplex protein metabolism with high sensitivity, resolution andbiocompatibility in a broad spectrum of live cells, tissues and animals.

Exemplary Materials and Methods

Stimulated Raman Scattering Microscopy.

An integrated laser (picoEMERALD with custom modification, AppliedPhysics & Electronics, Inc.) was used as the light source for both Pumpand Stokes beams. Briefly, picoEMERALD provides an output pulse train at1064 nm with 6 ps pulse width and 80 MHz repetition rate, which servesas the Stokes beam. The frequency-doubled beam at 532 nm is used tosynchronously Seed a picosecond optical parametric oscillator (OPO) toproduce a mode-locked pulse train (the idler beam of the OPO is blockedwith an interferometric filter) with 5-6 ps pulse width. The wavelengthof the OPO is tunable from 720 to 990 nm, which serves as the Pump beam.The intensity of the 1064 nm Stokes beam is modulated sinusoidally by abuilt-in electro-optic modulator (EOM) at 8 MHz with a modulation depthof more than 95%. The Pump beam is spatially overlapped with the Stokesbeam with a dichroic mirror inside picoEMERALD. The temporal overlapbetween Pump and Stokes pulse trains is ensured with a built-in delaystage and optimized by the SRS signal of pure dodecane liquid.

Pump and Stokes beams are coupled into an inverted laser-scanningmicroscope (FV1200MPE, Olympus) optimized for near IR throughput. A 60×water objective (UPlanAPO/IR, 1.2 N.A., Olympus) with high near IRtransmission is used for all cellular level imaging, and a 25× waterobjective (XLPlan N, 1.05 N.A., MP, Olympus) with both high near IRtransmission and large field of view is used for brain tissue and invivo imaging. The Pump/Stokes beam size is matched to fill theback-aperture of the objective. The forward going Pump and Stokes beamsafter passing through the sample are collected in transmission with ahigh N.A. condenser lens (oil immersion, 1.4 N.A., Olympus), which isaligned following Kohler illumination. A telescope is then used to imagethe scanning mirrors onto a large area (10 mm by 10 mm) Si photodiode(FDS1010, Thorlabs) to descan beam motion during laser scanning. Thephotodiode is reverse-biased by 64 V from a DC power supply to increaseboth the saturation threshold and response bandwidth. A high O.D.bandpass filter (890/220 CARS, Chroma Technology) is used to block theStokes beam completely and transmit the Pump beam only. The outputcurrent of the photodiode is electronically pre-filtered by an 8-MHzband-pass filter (KR 2724, KR electronics) to suppress both the 80 MHzlaser pulsing and the low-frequency contribution due to laser scanningacross the scattering sample. It is then fed into a radio frequencylock-in amplifier (HF2LI, Zurich instrument) terminated with 50 S2 todemodulate the stimulated Raman loss signal experienced by the Pumpbeam. The R-output of the lock-in amplifier is fed back into the analoginterface box (FV10-ANALOG) of the microscope.

For HeLa cell imaging and brain tissue imaging, the time constant of thelock-in amplifier is set for 8 μs, and the images are acquired by a 12.5μs pixel dwell time, corresponding to 3.3 s for a 512-by-512 pixelframe. For neurons and in vivo imaging of embryonic zebrafish and micelivers and intestines, the time constant is set to be 20 μs, and theimages are acquired by a 40 μs of pixel dwell time, corresponding to10.5 s for a 512-by-512 pixel frame. Laser powers after 60× IR objectiveused for cell imaging are: 100 mW for modulated Stokes beam and 112 mWfor the Pump beam at 2133 cm⁻¹, 2000 cm⁻¹ and 1655 cm⁻¹ channels; 50 mWfor modulated Stokes beam and 56 mW for Pump beam at 2940 cm⁻¹ and 2845cm⁻¹ channels. Laser powers after 25× objective used for tissue and invivo imaging are: 134 mW for modulated Stokes beam; 120 mW for the Pumpbeam of 2133 cm⁻¹, 2000 cm⁻¹ and 1655 cm⁻¹ channels; 67 mW for modulatedStokes beam and 60 mW for Pump beam at 2940 cm⁻¹ and 2845 cm⁻¹ channels.

Metabolic Incorporation of Deuterated Amino Acids.

For HeLa cells: cells are Seeded on a coverslip in a petri-dish with 2mL of regular medium for 20 h, and then replaced with D-AA medium (orgroup I and group II D-AA media) for designated amount of time. Thecoverslip is taken out to make an imaging chamber filled with PBS forSRS imaging. For hippocampal neurons, the dissociated neurons fromnewborn mice are Seeded for 10 days in regular Neurobasal A medium, andthen replaced with the corresponding D-AA medium for designated amountof time before imaging. For organotypic brain slice, 400 μm thick, P10mouse brain slices are cultured on Millicell-CM inserts (PICM03050,millipore) in 1 mL CD-MEM culture medium for 2 h, and then change to in1 mL CD-neurobasal a culture medium for another 28 h before imaging. Fordetailed recipe of D-AA media and in vivo labeling procedure inzebrafish and mice. (See Supporting Information). The experimentalprotocol for in vivo mice experiments (AC-AAAG2702) and zebrafishexperiments (AC-AAAD6300) were approved by Institutional Animal Care andUse Committee at Columbia University.

Spontaneous Raman Spectroscopy.

The spontaneous Raman spectra were acquired using a laser Ramanspectrometer (inVia Raman microscope, Ranishaw) at room temperature. A27 mW (after objective) 532 nm diode laser was used to excite the samplethrough a 50×, N.A. 0.75 objective (NPLAN EPI, Leica). The total dataacquisition was performed during 60 seconds using the WIRE, software.All the spontaneous Raman spectra have subtracted the PBS solution asbackground.

Image Progressing.

Images are acquired with FluoView scanning software and assigned coloror overlaid by ImageJ. Linear combination was processed with Matlab.Graphs were assembled with Adobe Illustrator.

CuIture Medium.

Regular HeLa cells medium was made of 90% DMEM medium (11965,invitrogen), 10% FBS (10082, invitrogen) and 1× penicillin/streptomycin(15140, invitrogen); regular hippocampal neuron medium was made ofNeurobasal A Medium (10888, Invitrogen), 1×B27 serum free supplement(17504, Invitrogen) and 0.5 mM glutamine (25030, Invitrogen).

Htt-mEos2 Plasmid Construct and Transfection.

mHtt94Q-mEos2 plasmid was constructed by replacing CFP gene sequence inpTreTight-Htt94Q-CFP plasmid (Addgene, 23966) with mEos2 gene sequencefrom pRSETa-mEos2 plasmid (Addgene, 20341). For transfection ofmHtt-mEos2 plasmid in HeLa cells, 4 μg mHtt94Q-mEos2 plasmid wastransfected using Transfection Reagent (FuGene, Promega).

Optimized Deuterium-Labeling Media 1) D-AA medium (CD-DMEM) for HeLacells: adapted from regular recipe of DMEM medium (11965, Invitrogen).The D-AA culture medium for HeLa cells was made with 90% CD-DMEM, 10%FBS (10082, invitrogen) and 1× penicillin/streptomycin (15140,invitrogen).

Amino acids Concentration Product company components (mM) and catalognumber Glycine-d₅ 0.4 DLM-280, Cambridge isotope L-Arginine•HCl-d₇ 0.398DLM-541, Cambridge isotope L-Cysteine•2HCl 0.2 C6727, SIGMA (regular)*L-Glutamine-d₅ 4.0 DLM-1826, Cambridge isotope L-Histidine•HCl•H₂O 0.2H5659, SIGMA (regular)* L-Isoleucine-d₁₀ 0.802 DLM-141, Cambridgeisotope L-Leucine-d₁₀ 0.802 DLM-567, Cambridge isotope L-Lysine•HCl-d₈0.798 616214, ALDRICH (Isotech) L-Methionine-d₃ 0.201 DLM-431, Cambridgeisotope L-Phenylalanine-d₈ 0.4 DLM-372, Cambridge isotope L-Serine-d₃0.4 DLM-582, Cambridge isotope L-Threonine 0.798 T8441, SIGMA (regular)*L-Tryptophan 0.078 T8941, SIGMA (regular)* L-Tyrosine-d₂ 0.398 DLM-2317,Cambridge isotope L-Valine-d₈ 0.803 DLM-488, Cambridge isotopeOther components (vitamins, Inorganic Salts and glucose) are exactly thesame as in the regular DMEM medium (11965, invitrogen).

* The reasons these 4 amino acids are remain in their regular forms arebecause: first, their deuterated forms have limited number of side chaindeuterium and are also relatively expensive; second, their occurrence(percentage) in mammalian cell proteins are small. Thus the lack of thedeuterated version for these 4 amino acids would not influence thegeneral deuterium labeling efficiency for CD-DMEM. Same reason appliesto below media.

2) D-AA medium (CD-Neurobasal A) for hippocampal neuron culture andorganotypic brain slices: adapted from regular recipe of Neurobasal Amedium (10888, Invitrogen). The D-AAs culture medium for hippocampalneurons was made of CD-Neurobasal A Medium, 1×B27 serum free supplement(17504, Invitrogen) and 0.5 mM glutamine-d₅ (DLM-1826, Cambridgeisotope). The CD-Neurobasal A culture medium for organotypic brainslices was made of CD-Neurobasal A Medium, 1×B27 serum free supplement(17504, Invitrogen), 0.5% glucose (15023, invitrogen), 2 mM glutamine-d5(DLM-1826, Cambridge isotope) and 1× penicillin/streptomycin (15140,invitrogen).

Amino acids Concentration Product company components (mM) and catalognumber Glycine-d₅ 0.4 DLM-280, Cambridge isotope L-Alanine-d₄ 0.022DLM-250, Cambridge isotope L-Arginine•HCl-d₇ 0.398 DLM-541, Cambridgeisotope L-Asparagine-d₈ 0.006 672947 ALDRICH (Isotech) L-Cysteine•2HCl0.26 C6727, SIGMA (regular)* L-Histidine•HCl•H₂O 0.2 H5659, SIGMA(regular)* L-Isoleucine-d₁₀ 0.802 DLM-141, Cambridge isotopeL-Leucine-d₁₀ 0.802 DLM-567, Cambridge isotope L-Lysine•HCl-d₈ 0.798616214, ALDRICH (Isotech) L-Methionine-d₃ 0.201 DLM-431, Cambridgeisotope L-Phenylalanine-d₈ 0.4 DLM-372, Cambridge isotope L-Proline-d₇0.067 DLM-487, Cambridge isotope L-Serine-d₃ 0.4 DLM-582, Cambridgeisotope L-Threonine 0.798 T8441, SIGMA (regular)* L-Tryptophan 0.078T8941, SIGMA (regular)* L-Tyrosine-d₂ 0.398 DLM-2317, Cambridge isotopeL-Valine-d₈ 0.803 DLM-488, Cambridge isotopeOther components (vitamins, Inorganic Salts and glucose) are exactly thesame as in the regular Neurobasal A medium (10888, Invitrogen).

3) Group I D-AA medium for HeLa cells. The group I D-AA culture mediumfor HeLa cells was made with 90% group I D-AA medium, 10% FBS (10082,invitrogen) and 1× penicillin/streptomycin (15140, invitrogen),

Concentration Product company Amino acids components (mM) and catalognumber Glycine 0.4 50046, SIGMA (regular) L-Arginine•HCl 0.398 A6969,SIGMA (regular) L-Cysteine•2HCl 0.2 C6727, SIGMA (regular) L-Glutamine4.0 G8540, SIGMA (regular) L-Histidine•HCl•H₂O 0.2 H5659, SIGMA(regular) L-Isoleucine-d₁₀ 0.802 DLM-141, Cambridge isotopeL-Leucine-d₁₀ 0.802 DLM-567, Cambridge isotope L-Lysine•HCl 0.798 L8662SIGMA (regular) L-Methionine 0.201 M5308 SIGMA (regular) L-Phenylalanine0.4 P5482 SIGMA (regular) L-Serine 0.4 S4311 SIGMA (regular) L-Threonine0.798 T8441, SIGMA (regular) L-Tryptophan 0.078 T8941, SIGMA (regular)L-Tyrosine 0.398 T8566 SIGMA (regular) L-Valine-d₈ 0.803 DLM-488,Cambridge isotopeOther components (vitamins, Inorganic Salts and glucose) are exactly thesame as in the regular DMEM medium (11965, invitrogen).

4) Group II D-AA medium for HeLa cells. The group II D-AA culture mediumfor HeLa cells was made with 90% group II D-AA medium, 10% FBS (10082,invitrogen) and 1× penicillin/streptomycin (15140, invitrogen).

Amino acids Concentration Product company components (mM) and catalognumber Glycine-d₅ 0.4 DLM-280, Cambridge isotope L-Arginine•HCl-d₇ 0.398DLM-541, Cambridge isotope L-Cysteine•2HCl 0.2 C6727, SIGMA (regular)L-Glutamine-d₅ 4.0 DLM-1826, Cambridge isotope L-Histidine•HCl•H₂O 0.2H5659, SIGMA (regular) L-Isoleucine 0.802 I7403 SIGMA (regular)L-Leucine 0.802 L8912 SIGMA (regular) L-Lysine•HCl-d₈ 0.798 616214,ALDRICH (Isotech) L-Methionine-d₃ 0.201 DLM-431, Cambridge isotopeL-Phenylalanine-d₈ 0.4 DLM-372, Cambridge isotope L-Serine-d₃ 0.4DLM-582, Cambridge isotope L-Threonine 0.798 T8441, SIGMA (regular)L-Tryptophan 0.078 T8941, SIGMA (regular) L-Tyrosine-d₂ 0.398 DLM-2317,Cambridge isotope L-Valine 0.803 V0513 SIGMA (regular)Other components (vitamins, Inorganic Salts and glucose) are exactly thesame as in the regular DMEM medium (11965, invitrogen).

5) D-AA medium (CD-MEM) for organotypic brain slice: adapted fromregular recipe of MEM medium (11095, Invitrogen). The CD-MEM culturemedium for organotypic brain slice was made with 90% CD-MEM, 10% FBS(10082, invitrogen) 0.5% glucose (15023, invitrogen) and 1×penicillin/streptomycin (15140, invitrogen).

Amino acids Concentration Product company components (mM) and catalognumber L-Arginine•HCl-d₇ 0.597 DLM-541, Cambridge isotopeL-Cysteine•2HCl 0.1 C6727, SIGMA (regular)* L-Glutamine-d₅ 2.0 DLM-1826,Cambridge isotope L-Histidine•HCl•H₂O 0.2 H5659, SIGMA (regular)*L-Isoleucine-d₁₀ 0.397 DLM-141, Cambridge isotope L-Leucine-d₁₀ 0.397DLM-567, Cambridge isotope L-Lysine•HCl-d₈ 0.399 616214, ALDRICH(Isotech) L-Methionine-d₃ 0.1 DLM-431, Cambridge isotopeL-Phenylalanine-d₈ 0.19 DLM-372, Cambridge isotope L-Threonine 0.403T8441, SIGMA (regular)* L-Tryptophan 0.049 T8941, SIGMA (regular)*L-Tyrosine-d₂ 0.199 DLM-2317, Cambridge isotope L-Valine-d₈ 0.393DLM-488, Cambridge isotopeOther components (vitamins, Inorganic Salts and glucose) are exactly thesame as in the regular MEM medium (11095, invitrogen).

6) For zebrafish: Wild-type zebrafish embryos at the 1-cell stage wereinjected with 1 nL D-AA solution and allowed to develop normally foranother 24 h. The zebrafish embryos at 24 hpf were manuallydechorionated before imaging. D-AA solution was made of 150 mg uniformlydeuterium-labeled amino acid mix (20 aa) (DLM-6819, Cambridge Isotope)dissolved in 1 mL PBS, with subsequent filtration using Milliporesterile syringe Filters (0.22 μm, SLGV033RS).

7) For mice: 1. Oral administration: 3-week-old mice were fed with D-AAcontaining drinking water for 12 days before harvesting the liver andintestine tissues. The drinking water was made of 500 mg uniformlydeuterium-labeled amino acid mix (20 aa) (DLM-6819, Cambridge Isotope)dissolved in 200 ml PBS, with subsequent filtration using Milliporesterile syringe Filters (0.22 μm, SLGV033RS). 2. Intraperitonealinjection: 3-week-old mice were injected with 500 μl D-AAs solution atthe 0^(th) h, 12^(th) h and 24^(th) h. The tissues were then harvestedat the 36^(th) h after the first injection. D-AA solution was made of500 mg uniformly deuterium-labeled amino acid mix (20 aa) (DLM-6819,Cambridge Isotope) dissolved in 2 ml PBS solutions, with subsequentfiltration using Millipore sterile syringe Filters (0.22 μm, SLGV033RS).

Example 1a: Sensitivity Optimization and Time-Lapse Imaging of the DeNovo Proteome Synthesis Dynamics

The cell culture medium reported previously was prepared by supplyinguniformly deuterium-labeled whole set of amino acids to a commerciallyavailable medium that is deficient of leucine, lysine and arginine (WeiL, Yu Y, Shen Y, Wang M C, Min W (2013) Vibrational imaging of newlysynthesized proteins in live cells by stimulated Raman scatteringmicroscopy. Proc. Natl. Acad. Sci. USA. 110:11226-11231). Due to thepresence of other regular amino acids already in the commercial medium,the resulting partially deuterated medium has only about 60% deuterationefficiency. In the present paper, we custom prepared new media thatreplace nearly all the regular amino acids by the D-AA counterparts(details in Supporting Information). As shown in the spontaneous Ramanspectra (FIG. 2 a ), the optimized medium (spectrum 205) displays a 50%signal increase compared with the partially deuterated medium (spectrum210). Indeed, SRS images targeting C-D vibrational peak at 2133 cm⁻¹confirms a 50% average intensity boost in live HeLa cells (FIG. 2 b ).The use of optimized D-AA medium now leads to an about 8 times highersignal than when using a single leucine-d₁₀ (FIG. 2 a , spectrum 205 vs.spectrum 210). In addition to improving labeling strategy, non-trivialinstrumentation optimizations are also carried out to further improveSRS detection sensitivity and acquisition speed, including increasingthe laser output and microscope system throughput for near-IRwavelengths, replacing the acousto-optic modulator (AOM) with anelectro-optic modulator (EOM) for a 30% higher modulation depth, andemploying a high-speed lock-in amplifier for faster image acquisition.

With much-improved sensitivity, protein synthesis can now be imaged withsuperb spatial and temporal resolutions. Spatially, we visualized newlysynthesized proteins from fine structures (likely dendritic spines,indicated by arrow heads) of live neurons (FIG. 2 c ). Temporally, wecould readily image newly synthesized proteins in live HeLa cells inless than one-hour incubation with the optimized deuteration medium(FIG. 2 d ). Control image with protein synthesis inhibitors onlydisplays vague cell outlines which presumably come from the free D-AApool (sub-mM concentration). Moreover, using a fast lock-in amplifier(details in Methods), our current imaging speed can be as fast as 3 sper frame (512×512 pixels), nearly 10 times faster than before, whichenables time-lapse imaging in live cells with minimum photo-toxicity tocell viabilities. FIG. 2 e presents time-lapse SRS imaging of a same setof live HeLa cells gradually synthesizing new proteins over time from 10min to 5 h incubation in optimized D-AA medium. The obvious cellmigration and division prove their viability, supporting highbio-compatibility of our technique. To our best knowledge, this is thefirst time that long-term time-lapse imaging of proteome synthesisdynamics is demonstrated on single live mammalian cells.

Example 1b: SRS Imaging of Protein Degradation in Live HeLa Cells

Besides imaging protein synthesis, our imaging platform offers theability to probe protein degradation simultaneously. Experimentally, weintend to probe the pre-existing protein pool by targeting the CH3showing a strong peak at 2940 cm-1, as newly synthesized proteins willbe mostly carrying C-D peaked around 2133 cm-1. However, the 2940 cm-1CH3 protein channel is known to suffer from undesired crosstalk from theCH2 lipid signal peaked at 2845 cm-1. To obtain a clean proteincomponent, we adopt two-color SRS imaging at both 2940 cm-1 and 2845cm-1 channels followed by a linear combination algorithm which has beeneffectively applied in cells, tissues and animals. The subsequentlyobtained images show the pure distribution of old protein pools(exclusively from CH3) and the distribution of lipids (exclusively fromCH2), respectively. Hence protein degradation could be tracked byimaging the old protein distributions over time when cells are growingin the D-AA medium.

FIG. 3 a shows time-dependent SRS images of old protein distributions(CH3) in live HeLa cells when incubated with D-AAs from 0 h to 96 h.Clearly, the old protein pool is degrading, as shown by the decay of itsaverage intensity. As a contrast, the total lipid images display noobvious intensity change (FIG. 3 b ). In addition, the spatial patternsof old proteins (FIG. 3 a ) reveal a faster decay in the nucleoli thanthe cytoplasm. This observation is consistent with the fact thatneucleoli have active protein turnover and also with our previous reportthat C-D labeled newly synthesized proteins are more prominent innucleoli (Wei L, Yu Y, Shen Y, Wang M C, Min W (2013) Vibrationalimaging of newly synthesized proteins in live cells by stimulated Ramanscattering microscopy. Proc. Natl. Acad. Sci. USA. 110:11226-11231).Single exponential decay fitting of the average intensities in FIG. 3 ayields a decay time constant of 45±4 h (FIG. 3 c ), corresponding to aproteome half-live of 31±3 h which is very close to the data reported bymass spectrometry (35 h) (Cambridge S B et al. (2011) Systems-wideproteomic analysis in mammalian cells reveals conserved, functionalprotein turnover. J. Proteome Res. 10:5275-5284). Therefore, our imagingplatform is capable of observing both protein synthesis and degradationby imaging at C-D channel and CH₃ channel, respectively, thus capturingproteomic metabolism dynamics in full-scope.

Retrieval of pure CH₃ and CH₂ signals by linear combination between 2940cm⁻¹ and 2845 cm⁻¹ channels was conducted employing equations follow LuF-K et al. (2012) Multicolor stimulated Raman scattering microscopy.Mol. Phys. 110:1927-1932; and Yu Z et al. (2012) Label-free chemicalimaging in vivo: three-dimensional non-invasive microscopic observationof amphioxus notochord through stimulated Raman scattering (SRS). Chem.Sci. 3:2646-2654. Pure CH₃ signal can be retrieved as[c]_(protein)∝5.2*(2940 cm⁻¹ signal)−4.16*(2845 cm⁻¹ signal); Pure CH₂signal can be retrieved as [c]_(lipid)∝1.2*(2845 cm⁻¹ signal)−0.3*(2940cm⁻¹ signal). This algorithm was tested with skin tissue samples,yielding similar results as reported in Lu F-K et al. (2012) Multicolorstimulated Raman scattering microscopy. Mol. Phys. 110:1927-1932; and YuZ et al. (2012) Label-free chemical imaging in vivo: three-dimensionalnon-invasive microscopic observation of amphioxus notochord throughstimulated Raman scattering (SRS). Chem. Sci. 3:2646-2654.

Example 1c: Two-Color Pulse-Chase SRS Imaging of Two Sets of TemporallyDefined Proteins

Inspried by the popular pulse-chase analysis in classic autoradiographytechniques and recent two-color BONCAT imaging (Beatty K E, Tirrell D A(2008) Two-color labeling of temporally defined protein populations inmammalian cells. Bioorg. Med. Chem. Lett. 18:5995-5999), we aim toexploit another dimension of probing dynamic protein metabolism withtwo-color pulse-chase imaging of proteins labeled at different times. Todo so, we need to rationally divide total D-AAs into two sub-sets withdistinct Raman spectra. We reasoned that Raman peaks of C-D stretchingare closely related to their chemical environments, thus the structuraldifference between D-AAs should lead to diverse Raman peak positions andshapes. We then examined the spontanesous Raman spectra of each D-AAsequentially, and subsequently identified two subgroups. Group Icontains three amino acids, leuine-d10, isoleucine-d10 and valine-d8,structurally known as branched-chain amino acids (FIG. 4 a ). Allmembers of group I exhibits multiple distinct Raman peaks with the firstone around 2067 cm-1.

The rest of D-AAs without branched chains are then categorized intogroup II, all of which show a prominent Raman peak around 2133 cm-1(three examples shown in FIG. 4 b ). To test inside cells, Raman spectraof HeLa cells cultured in either group I D-AA medium only (element 405)or group II D-AA medium only (element 410) are shown in FIG. 4 c . Basedon the spectra, we choose to acquire two-color narrow-band SRS images at2067 cm-1 and at 2133 cm-1. By constructing and utilizing a linearcombination algorithm, similar to the one used for CH₃ and CH₂ above,pure signals of proteins labeled by group I D-AAs and by group II D-AAscan be successfully separated and quantitatively visualized (e.g. FIGS.7 a-7 b ). In other exemplary embodiments, the images are obtained witha hyper-spectral imaging approach using broadband femtosecond lasers.

We now chose the mutant huntingtin (mHtt) protein in Huntington'sdisease as our model system for pulse-chase imaging demonstration. It isbelieved that Huntington's disease is caused by a mutation from normalhuntingtin gene to mHtt gene expressing aggregation-prone mHtt proteinswith poly-glutamine (polyQ) expansion (Walker FO (2007) Huntington'sDisease. The Lancet. 369: 218-228). For easy visualization byfluorescence, we tagged mHtt (with 94Q) with a fluorescent proteinmarker, mEos2. As illustrated by the cartoon in FIG. 9 d , HeLa cellswere first transfected with mHtt94Q-mEos2 plasmid in regular medium for4 h, and then replaced with group II D-AA medium for 22 h beforechanging to group I D-AA medium for another 20 h. SRS images areacquired at 2067 cm-1 and 2133 cm-1 channels, respectively, andsubsequently processed with linear combination.

Fluorescence overlaid with bright field image informs us the formationof a large aggregate triggered by aggregation-prone polyQ expansion inmHtt94Q-mEos2 (FIG. 4 d , fluorescence). Interestingly, proteins labeledwith group II D-AAs during the initial pulse period mainly concentratewithin the core of the aggregate (FIG. 4 d , element 415), whereasproteins labeled with group I D-AAs during the subsequent chase periodoccupy the entire volume of the aggregate (FIG. 4 d , element 420). Themerged image between group I and group II images, as well as theintensity profiles across the aggregate, further confirm the observationof a yellow core inside and a green shell outside (FIG. 4 d , merged).This two-color pulse-chase result suggests that the core is aggregatedearlier in time and the later produced mHtt proteins are then recruitedto and percolate through the aggregate to increases its overall size, inagreement with recently reported results by fluorescence (Schipper-KromS et al. (2014)) Dynamic recruitment of active proteasomes intopolyglutamine initiated inclusion bodies. FEBS Lett. 588:151-159). Thedemonstration here thus illustrates that our imaging platform using thetwo subgroups of D-AAs is readily applicable for performing pulse-chaseimaging to probe the complex and dynamic aspects of proteome metabolism.

In order to achieve SRS imaging of pure group I D-AA labeled proteindistribution and pure group II D-AA labeled protein distributionsimultaneously, we construct a robust linear combination algorithm toretrieve the underlying pure concentration information for two-colorpulse-chasing imaging similar to the one presented above from Lu F-K etal. (2012) Multicolor stimulated Raman scattering microscopy. Mol. Phys.110:1927-1932; and Yu Z et al. (2012) Label-free chemical imaging invivo: three-dimensional non-invasive microscopic observation ofamphioxus notochord through stimulated Raman scattering (SRS). Chem.Sci. 3:2646-2654. Since SRS signals exhibit linear concentrationdependence with analyte concentrations, two chemical species withdifferent Raman spectra can be retrieved quantitatively with two-colorSRS imaging. Hence, based on the spectra shown in FIG. 4 c , we chooseto acquire narrow-band SRS images at 2067 cm-1 and 2133 cm-1 channels,respectively, and perform subsequent linear combination algorithm toremove the spectral cross-talk.

The proper algorithm with the corresponding cross-talk coefficients isconstructed with SRS images of standard reference samples, i.e., puregroup I D-AA labeled protein and pure group II D-AA labeled protein. Todo so, we labeled HeLa cells with only group I D-AA medium FIG. 7 a andonly group II D-AA medium FIG. 7 b , respectively, and acquired a set ofimage pairs at 2067 cm-1 and 2133 cm-1 channels for each cell samples.

For any sample labeled with both groups of D-AAs, the measured SRSsignals at 2067 cm-1 and 2133 cm-1 channels can be written as thefollowing, with linear relationship to group I D-AA and group II D-AAconcentrations ([c]group I and [c]group II):

${\begin{bmatrix}{2067{cm}^{- 1}{signal}} \\{2133{cm}^{- 1}{signal}}\end{bmatrix} = {\begin{bmatrix}i_{{groupI},{2067{cm}^{- 1}}} & i_{{groupII},{2067{cm}^{- 1}}} \\i_{{groupI},{2133{cm}^{- 1}}} & i_{{groupII},{2133{cm}^{- 1}}}\end{bmatrix}\begin{bmatrix}\lbrack c\rbrack_{groupI} \\\lbrack c\rbrack_{groupII}\end{bmatrix}}},$

where i_(groupI,2067cm) ⁻¹ , i_(groupI,2133cm) ⁻¹ , i_(groupII,2067cm)⁻¹ , i_(groupII,2133cm) ⁻¹ are the average pixel intensity recordedinside cells in FIG. 7 a and FIG. 7 b.

Thus group I D-AA and group II D-AA concentrations can then be easilysolved as:

${\lbrack c\rbrack_{g{roupI}} = \frac{\begin{matrix}{{i_{{groupII},{2133{cm}^{- 1}}}\left( {2067{cm}^{- 1}{signal}} \right)} -} \\{i_{{groupII},{2067{cm}^{- 1}}}\left( {2133{cm}^{- 1}{signal}} \right)}\end{matrix}}{{i_{{groupII},{2133{cm}^{- 1}}}i_{{groupI},{2067{cm}^{- 1}}}} - {i_{{groupII},{2067{cm}^{- 1}}}i_{{groupI},{2133{cm}^{- 1}}}}}},$$\lbrack c\rbrack_{groupII} = {\frac{\begin{matrix}{{i_{{groupI},{2067{cm}^{- 1}}}\left( {2133{cm}^{- 1}{signal}} \right)} -} \\{i_{{groupI},{2133{cm}^{- 1}}}\left( {2067{cm}^{- 1}{signal}} \right)}\end{matrix}}{{i_{{groupII},{2133{cm}^{- 1}}}i_{{groupI},{2067{cm}^{- 1}}}} - {i_{{groupII},{2067{cm}^{- 1}}}i_{{groupI},{2133{cm}^{- 1}}}}}.}$

Taking the average pixel intensity recording in FIG. 7 a and FIG. 7 binto the above equations, the final linear combination algorithm readsas:

[c]group I∝1.06*(2067 cm-1 signal)−0.0047*(2133 cm-1 signal),  (1)

[c]groupII∝(2133 cm-1 signal)−1.15*(2067 cm-1 signal).  (2)

Example 1d: SRS Imaging of Newly Synthesized Proteins in Live MouseBrain Tissues

Going above the cellular level, we now apply our imaging platform to amore complex level, organotypical brain tissues. In our study, we focuson the hippocampus because it is the key region in brains that involvesextensive protein synthesis. As expected, active protein synthesis isfound in the hippocampal region, particularly in the dentate gyrus,which is known for its significant role in both long-term memoryformation and adult neurogenesis. SRS image at 2133 cm-1 (FIG. 5 a ,C-D) of a live mouse organotypic brain slices cultured in D-AA mediumfor 30 h, reveals active protein synthesis from both the soma and theneurites of individual neurons in dentate gyrus. In addition, the oldprotein (CH₃) and total lipids (CH₂) images are presented simultaneouslyfor multichannel analysis (FIG. 5 a ).

In order to investigate spatial pattern of protein synthesis on a largerscale, we imaged the entire brain slice by acquiring large-area imagemosaics. A 4-by-3 mm image (FIG. 5 b ) of another organotypic slicedisplays overlayed patterns from new proteins (2133 cm-1, element 505),old proteins (CH₃, element 510) and lipids (CH₂, element 515).Intriguing spatial variation is observed: while the distribution of oldproteins are relatively homogenous across the field of view, newlysynthesized proteins are either concentrated in dentate gyrus orscattered within individual neurons throughout the cortex, suggestinghigh activities in these two regions. Thus, we have demonstrated theability to directly image protein synthesis dynamics on living braintissues with subcellular resolution and multi-channel analysis, whichwas difficult to achieve with other existing methods. The intricaterelationship between protein synthesis and neuronal plasticity iscurrently under investigation on this platform.

Example 1e: SRS Imaging of Newly Synthesized Proteins In Vivo

One prominent advantage of our labeling strategy is its non-toxicity andminimal invasiveness to animals. We thus move up to the physiologicallevel to image protein metabolism in embryonic zebrafish and mice.Zebrafish are popular model organisms due to their well-understoodgenetics and transparent embryos, amenable to optical imaging. Weinjected 1 nL D-AA solution into zebrafish embryos at the 1-cell stage(150 ng D-AAs per embryo), and then allowed them to develop normally for24 h (FIG. 6 a , bright field) before imaging the whole animal. We founda high signal of newly synthesized proteins (FIG. 6 a, 2133 cm-1) in thesomites at the embryonic zebrafish tail, consistent with the earlierBONCAT result (Hinz F I, Dieterich D C, Tirrell D A, Schuman E M (2012)Non-canonical amino acid labeling in vivo to visualize and affinitypurify newly synthesized proteins in larval zebrafish. ACS Chem.Neurosci. 3:40-49). The spatial pattern of this signal appears similarto that of the old protein distribution (FIG. 6 a , CH₃), but almostcomplementary to the lipid distribution (FIG. 6 a , CH2).

Finally we demonstrate on mammals—mice. We administered the drinkingwater containing D-AAs to 3-week-old mice for 12 days, and thenharvested liver and intestine tissues for subsequent imaging. Notoxicity was observed for the fed mice. The SRS images from both liveliver tissues (FIG. 6 b ) and live intestine tissues (FIG. 6 c )illustrate the distributions of newly synthesized proteins (2133 cm-1,C-D) during the feeding period, which resemble the total proteindistribution (1655 cm-1, Amide I). On a faster incorporation timescale,live liver and intestine tissues obtained after intraperitonealinjection of D-AAs into mice for 36 h reveal spatial patterns (FIGS. 6b-6 c ) similar to the feeding results above as well as theclick-chemistry based fluorescence staining (Liu J, Xu Y, Stoleru D,Salic A (2012) Imaging protein synthesis in cells and tissues with analkyne analog of puromycin. Proc. Natl. Acad. Sci. USA 109:413-418). Allthese results support our imaging platform as a highly suitabletechnique for in vivo interrogation.

FIGS. 8 a-8 c show raw C-D on-resonance (2133 cm-1) and off-resonance(2000 cm-1) SRS images of newly synthesized proteins in vivo in FIGS. 6a-6 c . FIG. 8 a SRS C-D on-resonance and off-resonance images of a 24hpf embryonic zebrafish. The difference image between C-D on-resonanceand off-resonance (pixel-by-pixel subtraction) shows pure C-D labeledprotein distribution in the somites of an embryonic zebrafish tail, asin FIG. 6 a . FIGS. 8 b-8 c SRS C-D on-resonance and off-resonanceimages of live mouse liver FIG. 8 b and intestine FIG. 8 c tissuesharvested from the mice after administering with D-AA containingdrinking water for 12 days. The difference image between C-Don-resonance and off-resonance (pixel-by-pixel subtraction) shows pureC-D labeled protein distribution in the liver and intestine tissues,shown in FIG. 6 b and FIG. 6 c , respectively. The residual signalpresented in the off-resonance images mainly comes from cross-phasemodulation induced by highly scattering tissue structures.

FIGS. 9 a-9 d show SRS imaging for newly synthesized proteins in vivowith intraperitoneal injection of mice with D-AA solutions. FIGS. 9 a-9b SRS images of live mouse liver FIG. 9 a and intestine 9b tissuesharvested from mice after intraperitoneal injection injected with D-AAssolutions for 36 h. 2133 cm-1 channel shows newly synthesized proteins(off-resonance image subtracted) that resemble the distribution of totalproteins as shown in the 1655 cm-1 image (Amide I). (c-d) Correspondingraw C-D on-resonance (2133 cm-1) and off-resonance (2000 cm-1) imagesare shown as references for liver c and intestine d tissues. Scale bar,10 μm.

Exemplary Physical Principle of Isotope-Based SRS Imaging

SRS microscopy can be a molecular-contrast, highly sensitive, imagingprocedure with intrinsic 3D sectioning capability. It selectively imagesthe distribution of molecules that carry a given type of chemical bondsthrough resonating with the specific vibrational frequency of thetargeted bonds. (See, e.g., References 47, 54 and 65). As FIG. 5 aillustrates, by focusing both temporally and spatially overlapped Pumpand Stokes laser pulse trains into samples, the rate of vibrationaltransition can be greatly amplified by about 107 times when the energydifference of the two laser beams matches the particular chemical bondvibration, Ωvib. (See, e.g., Reference 65). Accompanying such stimulatedactivation of one vibrational mode, one photon can be created into theStokes beam, and simultaneously another photon can be annihilated fromthe Pump beam, a process called stimulated Raman gain and stimulatedRaman loss, respectively. The energy difference between the Pump photonand the Stokes photon can be used to excite the vibrational mode,fulfilling energy conservation. FIG. 5 b shows a high-frequencymodulation procedure, where the intensity of the Stokes beam can beturned on and off at 10 MHz, and can be employed to achieveshot-noise-limited detection sensitivity by suppressing laser intensityfluctuations occurring at low frequencies. The transmitted Pump beamafter the sample can be detected by a large-area photodiode, and thecorresponding stimulated Raman loss signal, which also occurs at 10 MHz,can be demodulated by a lock-in amplifier. By scanning across the samplewith a laser-scanning microscope, a quantitative map with chemicalcontrast can be produced from the targeted vibrating chemical bonds. Asthe SRS signal can be dependent on both Pump and Stokes laser beams, thenonlinear nature can provide a 3D optical sectioning ability.

The vibrational signal of C-D can be detected as an indicator for newlysynthesized proteins that metabolically incorporate deuterium-labeledamino acids. (See, e.g., FIG. 5 b ). When hydrogen atoms can be replacedby deuterium, the chemical and biological activities of biomoleculesremain largely unmodified. The C-D stretching motion can display adistinct vibrational frequency from all the other vibrations ofbiological molecules inside live cells. The reduced mass of the C-Doscillator can be increased by two folds when hydrogen can be replacedby deuterium. Based on the above Equation, Ωvib can be reduced by afactor of 2. The experimentally measured stretching frequency can beshifted from ˜2950 cm-1 of C-H to ˜2100 cm-1 of C-D. The vibrationalfrequency of 2100 cm-1 can be located in a cell-silent spectral windowin which no other Raman peaks exist, thus enabling detection ofexogenous C-D with both high specificity and sensitivity.

Imaging optimization by metabolic incorporation of deuterium-labeled allamino acids in live HeLa cells with multicolor SRS imaging. Althoughleucine can be the most abundant essential amino acid, it only accountsfor a small fraction of amino acids in proteins. Thus, the deuteriumlabeling of all the amino acids can lead to a substantial signalenhancement. Indeed, the spontaneous Raman spectrum (e.g., FIG. 33 a )of HeLa cells incubated with deuterium-labeled all 20 amino acids (e.g.,prepared by supplying uniformly deuterium-labeled whole set of aminoacids to leucine, lysine and arginine deficient DMEM medium) can exhibitC-D vibrational peaks about five times higher than shown in FIG. 2 aunder the same condition. The corresponding SRS image at 2133 cm-1(e.g., FIG. 33 b ) can show a significantly more pronounced signal thanthat in FIG. 2 a under the same intensity scale. In particular, nucleoli(e.g., indicated by arrows in FIG. 33 b and verified by DICvisualization) can exhibit the highest signal. Nucleoli, the activesites for ribosomal biogenesis, have been reported to involve rapidnucleolar assembly and proteomic exchange (See, e.g., Reference 68-70).Such fast protein turnover can be reflected by the spatial enrichment ofnewly synthesized protein signals in those subcellular areas. (See,e.g., FIG. 33 b ). Note that SRS imaging here can be directly performedon live cells and hence free from potential complications due tofixation and dye conjugation. Again, the off-resonant image at 2000 cm-1can be clean and dark (e.g., FIG. 33 c ), proving the specificity of SRSimaging of C-D at 2133 cm-1. In addition to imaging newly synthesizedproteins, SRS can readily image intrinsic biomolecules in a label-freemanner. By simply adjusting the energy difference between the Pump andthe Stokes beams to match the vibrational frequency of amide I, lipidsand total proteins respectively. FIGS. 33 d-33 f show the SRS images ofamide I band at 1655 cm-1 primarily attributed to proteins, CH₂stretching at 2845 cm-1 predominantly for lipids and CH₃ stretching at2940 cm-1 mainly from proteins with minor contribution from lipids.

Exemplary Time-Dependent De Novo Protein Synthesis and Protein SynthesisInhibition

Being linearly dependent on analyte concentration, SRS contrast can bewell suited for quantification of de novo protein synthesis in livecells. Here the time-dependent protein synthesis images can be shownunder the same intensity scale. (See, e.g., FIGS. 34 a-34 c ). Asexpected, the new protein signal (e.g., 2133 cm-1) from 5-hour, 12-hourand 20-hour incubation can increase substantially over time (e.g., FIGS.34 a-34 c ) while the amide I (e.g., 1655 cm-1) signal can remain at asteady state. (See, e.g., FIGS. 34 d-34 f ). Since protein distributioncan often be heterogeneous in biological systems, a more quantitativerepresentation by acquiring ratio images can be shown between the newlysynthesized proteins and the total proteome (e.g., from either amide Ior CH₃). FIGS. 34 g-34 i depict the fraction of newly synthesizedproteins (e.g., 2133 cm-1) among the total proteome (e.g., 1655 cm-1)and its spatial distribution. The fraction of newly synthesized proteinsgrowing with time from 5 hours to 20 hours can highlight nucleoli as thesubcellular compartments with fast protein turnover. (See, e.g.,Reference 68-70). Such quantitative ratio imaging of new versus oldproteomes can be very difficult to obtain using BONCAT or massspectroscopy without the destruction of cells. Moreover, FIG. 34 j showstime-lapse SRS images of a live dividing HeLa cell after 20-hourincubation in deuterium-labeled all amino acids medium, clearly provingthe viability of cells under the imaging condition.

The effect of protein synthesis inhibition by chemical drugs can befurther tested to validate that the detected C-D signal indeed derivesfrom nascent proteins. HeLa cells incubated with deuterium-labeled allamino acids together with 5 μM anisomycin, which can work as a proteinsynthesis inhibitor by inhibiting peptidyl transferase or the 80Sribosome system, show the absence of the C-D signal in the spontaneousRaman spectrum. (See, e.g., FIG. 34 k ). Furthermore, SRS imaging of thesame samples (e.g., FIG. 34 i ) can exhibit drastically weaker signal(See, e.g., Reference 71) when compared to FIG. 34 b without the proteinsynthesis inhibitor. As a control, the corresponding 2940 cm-1 image(e.g., FIG. 34 m ) of total proteome remains at a similar level as thenon-drug treated counterpart in FIG. 34 f Thus, the detected C-D SRSsignal (e.g., FIGS. 34 a-34 c ) can originate from deuterium-labelednascent proteins, which can vanish upon adding the protein synthesisinhibitor.

Exemplary Demonstration on HEK293T Cells and Neuron-Like DifferentiableNeuroblastoma N2A Cells

Two additional mammalian cell lines can be chosen for furtherdemonstration: human embryonic kidney HEK293T cells, and neuron-likeneuroblastoma mouse N2A cells, which can be induced to differentiatewith the growth of neurites (e.g., axons and dendrites). The spontaneousRaman spectrum (e.g., FIG. 35 a ) of HEK293T cells incubated withdeuterium-labeled all amino acids for 12 hours can exhibit a 2133 cm-1C-D channel signal nearly as high as the 1655 cm-1 amide channel signal.The resulting SRS image can show a bright signal for new proteins withan intense pattern residing in nucleoli. (See, e.g., FIG. 35 b ). Asbefore, the off-resonant image (e.g., 2000 cm-1) can display vanishingbackground (e.g., FIG. 35 c ); the amide I channel (e.g., 1655 cm-1)image (e.g., FIG. 35 d ) can exhibit consistent overall proteomedistributions similar to that in HeLa cells; CH₂ channel (e.g., 2845cm-1) image (e.g., FIG. 35 e ) depicts a more diffusive lipiddistribution in cytoplasm compared to that in HeLa cells. Consistentwith the results obtained in HeLa cells above, the ratio image (e.g.,FIG. 35 f ) between the newly synthesized proteins (e.g., FIG. 35 b )and the total proteins (e.g., FIG. 35 d ) highlights nucleoli for activeprotein turnover in HEK293T cells as well. (See, e.g., References44-46).

In addition to showing the ability to image newly synthesized proteinsinside cell body, the exemplary SRS can also be applied to tackle morecomplex problems, such as de novo protein synthesis in neuronal systems.(See, e.g., Reference 26-28). Under differentiation condition, N2A cellsmassively grow new neurites from cell bodies and form connections withother cells. FIG. 10 a shows the image of newly synthesized proteinsafter induction for differentiation, by simultaneously differentiatingthe N2A cells and supplying with the deuterium labeled all amino acidsfor 24 hours. Similar to HeLa and HEK293T cells, N2A cell bodies can beobserved to display high-level protein synthesis. Newly synthesizedproteins can also be observed in a subset of, but not all neurites(e.g., FIGS. 36 a and 36 b ), which can imply that the observed neuritesin FIG. 36 a can be newly grown under the differentiation condition. Fora detailed visualization, FIGS. 36 c and 10 d show the zoomed-in regionsin the dashed squares in FIGS. 36 a and 36 b respectively. A morecomprehensive examination is illustrated by both the ratio image (e.g.,FIG. 36 e ) between FIGS. 36 c and 36 d and the merged image (e.g., FIG.360 with 3605 designating new protein signal from FIGS. 36 c and 3610designating total protein signal from FIG. 36 d . On one hand, both theratio image and the merged image highlight the neurites with higherpercentage of new proteins (e.g., indicated by stars), implying theseneurites can be newly grown. On the other hand, from the merged image,there can be some neurites (e.g., indicated by arrows) showing obvioussignals in the green channel (e.g., total proteins) only but with nodetectable signal in the red channel (e.g., new proteins).

Hence, the neurites indicated by arrows can be most likely older thantheir starred counterparts. In addition, the transition from 3610 to3605 in the merged image (e.g., FIG. 36 f ) can imply the growthdirection by which new neurites form and grow. A more relevant system tostudy de novo protein synthesis and neuronal activities can behippocampal neurons, which can be known to be involved in long-termmemory formation (See, e.g., Reference 26-28). SRS image (e.g., 2133cm-1) of hippocampal neuron cells incubated with deuterium-labeled allamino acids can show a newly synthesized protein pattern in theneurites.

Example 2: In Vitro and In Vivo Labeling with Alkyne Tags ExemplaryMethods and Materials

Bond-Selective Stimulated Raman Scattering (SRS) Microscopy.

FIG. 37 b shows details of the microscopy setup. An integrated lasersystem (picoEMERALD, Applied Physics & Electronics, Inc.) was chosen asthe light source for both pump and Stokes beams. Briefly, picoEMERALDprovides an output pulse train at 1064 nm with 6 ps pulse width and 80MHz repetition rate, which serves as the Stokes beam. The frequencydoubled beam at 532 nm is used to synchronously Seed a picosecondoptical parametric oscillator (OPO) to produce a mode-locked pulse trainwith 5-6 ps pulse width (the idler beam of the OPO is blocked with aninterferometric filter). The output wavelength of the OPO is tunablefrom 720 to 990 nm, which serves as the pump beam. The intensity of the1064 nm Stokes beam is modulated sinusoidally by a built-inelectro-optic modulator at 8 MHz with a modulation depth of more than95%. The pump beam is then spatially overlapped with the Stokes beam byusing a dichroic mirror inside picoEMERALD. The temporal overlap betweenpump and Stokes pulse trains is ensured with a built-in delay stage andoptimized by the SRS signal of pure dodecane liquid at the microscope.

Pump and Stokes beams are coupled into an inverted multiphotonlaser-scanning microscope (FV1200MPE, Olympus) optimized for near-IRthroughput. A 60× water objective (UPlanAPO/IR, 1.2 N.A., Olympus) withhigh near-IR transmission is used for all cell imaging. The pump/Stokesbeam size is matched to fill the back-aperture of the objective. Theforward going pump and Stokes beams after passing through the sample arecollected in transmission with a high N.A. condenser lens (oilimmersion, 1.4 N.A., Olympus) which is aligned following Köhlerillumination. A telescope is then used to image the scanning mirrorsonto a large area (10 by 10 mm) Si photodiode (FDS1010, Thorlabs) todescan beam motion during laser scanning. The photodiode is reversebiased by 64 V from a DC power supply to increase both the saturationthreshold and response bandwidth. A high O.D. bandpass filter (890/220CARS, Chroma Technology) is placed in front of the photodiode to blockthe Stokes beam completely and to transmit the pump beam only.

The output current of the photodiode is electronically pre-filtered byan 8-MHz band-pass filter (KR 2724, KR electronics) to suppress both the80 MHz laser pulsing and the low-frequency fluctuations due to laserscanning cross the scattering sample. It is then fed into a radiofrequency lock-in amplifier (SR844, Stanford Research Systems)terminated with 50 S2 to demodulate the stimulated Raman loss signalexperienced by the pump beam. The in-phase X-output of the lock-inamplifier is fed back into the analog interface box (FV10-ANALOG) of themicroscope. The time constant is set for 10 μs (the shortest availablewith no additional filter applied). The current SRS imaging speed islimited by the shortest time constant available from the lock-inamplifier (SR844). For all imaging, 512 by 512 pixels are acquired forone frame with a 100 μs of pixel dwell time (26 s per frame) for laserscanning and 10 μs of time constant from the lock-in amplifier. Laserpowers after 60× IR objective used for imaging are: 130 mW for modulatedStokes beam; 120 mW for the pump beam in 2133 cm-1, 2142 cm-1, 2000 cm-1and 1655 cm-1 channels, 85 mW for the pump beam in 2230 cm-1 and 2300cm⁻¹ channels, and 50 mW for pump beam in 2845 cm⁻¹ channels.

Spontaneous Raman Spectroscopy.

The spontaneous Raman spectra were acquired using a laser confocal Ramanmicroscope (Xplora, Horiba Jobin Yvon) at room temperature. A 12 mW(after the microscope objective), 532 nm diode laser was used to excitethe sample through a 50×, N.A.=0.75 air objective (MPlan N, Olympus).The total data acquisition time was 300 s using the LabSpec 6 software.All the spontaneous Raman spectra have subtracted the PBS solutionbackground.

Materials.

5-Ethynyl-2′-deoxyuridine (EdU) (T511285), 17-Octadecynoic acid(17-ODYA) (08382), DMEM medium without L-methionine, L-cystine andL-glutamine (D0422), L-methionine (M5308), L-cystine (C7602),2-Mercaptoethanol (M3148) and Phorbol 12-myristate 13-acetate (P1585)were purchased from Sigma-Aldrich. 5-Ethynyl Uridine (EU) (E-10345),Homopropargylglycine (Hpg) (C10186), Alexa Fluor® 488 Azide (A10266),Click-iT® Cell Reaction Buffer Kit (C10269), DMEM medium (11965), FBS(10082), penicillin/streptomycin (15140), L-glutamine (25030),Neurobasal A Medium (10888) and B27 supplement (17504) were purchasedfrom Invitrogen. RPMI-1640 Medium (30-2001) was purchased from ATCC. BCS(hyclone SH30072) was purchased from Fisher Scientific.

DMEM culture medium was made by adding 10% (vol/vol) FBS and 1%(vol/vol) penicillin/streptomycin to the DMEM medium.Methionine-deficient culture medium was made by supplying 4 mML-glutamine, 0.2 mM L-cystine, 10% FBS and 1% penicillin/streptomycin tothe DMEM medium without L-methionine, L-cystine and L-glutamine.RPMI-1640 culture medium was made of supplying the RPMI-1640 medium with10% FBS, 1% penicillin/streptomycin and 50 μM 2-Mercaptoethanol. Neuronculture medium was made of Neurobasal A Medium adding with 1×B27supplement and 0.5 mM glutamine. CuIture medium for NIH3T3 cells wasmade by adding 10% (vol/vol) BCS and 1% (vol/vol)penicillin/streptomycin to the DMEM medium.

Propargylcholine Synthesis.

Propargylcholine was synthesized according to Jao, C. Y., Roth, M.,Welti, R. & Salic, A. Proc. Natl. Acad. Sci. USA 106, 15332-15337(2009). 3 mL propargyl bromide (80 wt. % solution in toluene) were addeddropwise to 3 g 2-dimethylaminoethanol in 10 mL anhydrous THF on iceunder argon gas protection and stirring. The ice bath was removed andthe mixture was kept stirring at room temperature overnight. The whitesolids were filtered the next day and washed extensively with coldanhydrous THF to obtain 5 g pure propargylcholine bromide. All chemicalshere are purchased from Sigma-Aldrich. NMR spectrum was recorded on aBruker 400 (400 MHz) Fourier Transform (FT) NMR spectrometers at theColumbia University Chemistry Department. ¹H NMR spectra are tabulatedin the following order: multiplicity (s, singlet; d, doublet; t,triplet; m, multiplet), number of protons. ¹H NMR (400 MHz, D₂O) δ ppm:4.37 (d, J=2.4 Hz, 2H); 4.10 (m, 2H); 3.66 (t, J=4.8 Hz, 2H); 3.28 (s,6H); MS (APCI+) m/z Calcd. for C₇H₁₄NO [M]⁺:128.19. Found: 128.26.

Sample Preparation for SRS Imaging of Live Cells and Organisms.

For all SRS imaging experiments of HeLa cells (e.g. FIG. 12 ), cellswere first Seeded on coverslips with a density of 1×10⁵/mL in petridishes with 2 mL DMEM culture medium for 20 h at 37° C. and 5% CO₂.

-   -   1) EdU experiment, DMEM culture medium was then changed to DMEM        medium (FBS-free) for 24 h for cell cycle synchronization. After        synchronization, medium was replaced back to DMEM culture medium        and EdU (10 mM stock in PBS) was simultaneously added to a        concentration of 100 μM for 15 h.    -   2) EU experiment, EU (100 mM stock in PBS) was added to the DMEM        culture medium directly to a concentration of 2 mM for 7 h.    -   3) Hpg experiment, DMEM culture medium was then changed to        methionine-deficient culture medium for 1 h, followed by        supplying 2 mM Hpg (200 mM stock in PBS) in the medium for 24 h.    -   4) Propargylcholine and EdU dual-color experiment, DMEM culture        medium was changed to DMEM medium (FBS-free) for        synchronization. After synchronization, medium was replaced back        to DMEM culture medium by simultaneously adding both        propargylcholine (25 mM stock in PBS) and EdU (10 mM stock in        PBS) to the culture medium to a concentration of 1 mM and 100        μM, respectively, for 24 h.

For the propargylcholine experiment in neurons, hippocampal neurons werecultured on coverslips in 1 ml neuron culture medium for 14 d, and thenpropargylcholine (25 mM stock in PBS) is directly added into the mediumto a final concentration of 1 mM for 24 h.

For the 17-ODYA experiment in macrophages, THP-1 cells were first Seededon coverslips at a density of 2×105/mL in 2 ml RPMI-1640 culture mediumfor 24 h, followed by 72 h induction of differentiation to macrophagesby incubating with 100 ng/ml Phorbol 12-myristate 13-acetate (PMA) inthe medium. Medium was then replaced with RPMI-1640 culture mediumcontaining 400 μM 17-ODYA (6:1 complexed to BSA) for 15 h.

For all of the above experiments, after incubation, the coverslip istaken out to make an imaging chamber filled with PBS for SRS imaging.

For the 17-ODYA experiment in C. elegans, OP50 bacterial culture wasmixed well with 4 mM 17-ODYA (from 100 mM ethanol stock solution), andthen Seeded onto nematode growth media (NGM) plates. After drying theplates in hood, wild type N2 day 1 adult C. elegans were placed onto theplates and fed for 40 h. C. elegans were then mounted on 2% agarose padscontaining 0.1% NaN3 as anesthetic on glass microscope slides for SRSimaging.

SRS Imaging of C. elegans Germline after Feeding with EdU.

MG1693 (thymidine defective MG1655) E. Coli strain was cultured in 2 mlLB medium at 37° C. overnight, and transferred to 100 ml of M9 mediumcontaining 400 μM EdU for further growth at 37° C. for 24 h. TheEdU-labeled MG1693 E. Coli was then Seeded on M9 agar plate.Synchronized day 1 adult worms developed in 20° C. were transferred toEdU-labeled bacterial plate for 3 h, and then were dissected to take outthe germline for imaging (e.g. FIG. 13 ).

Cell Preparation for Click Chemistry-Based Fluorescence Microscopy.

All experiments (e.g. FIGS. 15 a-15 c ) were carried out following themanufacturer's protocol from Invitrogen. HeLa cells were first incubatedwith 10 μM EdU in DMEM culture medium for 24 h, or 1 mM EU in DMEMculture medium for 20 h, or 1 mM Hpg in methionine-deficient culturemedium for 20h, respectively. Cells were then fixed in 4% PFA for 15min, washed twice with 3% BSA in PBS, permeabilized with 0.5% Triton PBSsolution for 20 min, and performed click chemistry staining using AlexaFluor 488 Azide in the Click-iT Cell Reaction Buffer Kit for 30 min.After washing with 3% BSA in PBS for three times, fluorescence imageswere obtained using an Olympus FV1200 confocal microscope with 488 nmlaser excitation while the cells were immersed in PBS solution.

Enzymatic Assays Confirming Propargylcholine Incorporation into CellularCholine Phospholipids.

We design our control experiments according to the click chemistry basedassays reported in Jao, C. Y., Roth, M., Welti, R. & Salic, A. Proc.Natl. Acad. Sci. USA 106, 15332-15337 (2009) (e.g. FIGS. 16 a-16 c ).NIH 3T3 cells cultured with 0.5 μM propargylcholine for 48 hours werefixed with 4% PFA for 15 minutes, rinsed with 1 mL TBS buffer twice andincubated with 1 mL 1 mg/mL BSA in TBS buffer for 1 hour at 37° C., withor without 0.02 U/mL phospholipase C (Type XIV from Clostridiumperfringens, Sigma), in the presence of 10 mM CaCl₂ (required forphospholipase C activity) (e.g. FIG. 16 b ) or 10 mM EDTA (e.g. FIG. 16c ). The cells were then washed with TBS buffer and ready for SRSimaging.

Sample Preparation for Drug Delivery into Mouse Ear Tissues.

Either DMSO solution or Drug cream (Lamisil, Novartis) containing 1%(w/w) active terbinafine hydrochloride (TH) was applied to the ears ofan anesthetized live mouse (2-3 weeks old white mouse of either sex) for30 min, and the dissected ears from the sacrificed mouse were thenimaged by SRS (e.g. FIGS. 17 b-17 e and FIGS. 18 a and 18 b ). The amide(1655 cm⁻¹) and lipid (2845 cm⁻¹) images have been applied with linearspectral unmixing to eliminate cross talk before composition. Theexperimental protocol for drug delivery on mice (AC-AAAG4703) wasapproved by Institutional Animal Care and Use Committee at ColumbiaUniversity.

Image Progressing.

Images are acquired with FluoView scanning software and assigned coloror overlaid by ImageJ. Graphs were assembled with Adobe Illustrator.

Example 2a: Alkyne Tags

As an effective imaging modality for small biomolecules, we report ageneral strategy of using stimulated Raman scattering (SRS) microscopyto image alkynes (i.e., C═C) as nonlinear vibrational tags, shown asbond-selective SRS in FIGS. 10 a-10 c . As shown in FIG. 10 a ,Spontaneous Raman spectra of HeLa cells and 10 mM EdU solution. Inset:the calculated SRS excitation profile (FWHM 6 cm⁻¹, element 905) is wellfitted within the 2125 cm⁻¹ alkyne peak (FWHM 14 cm⁻¹, magenta). FIG. 10b shows linear dependence of stimulated Raman loss signals (2125 cm⁻¹)with EdU concentrations under a 100 us acquisition time. FIG. 10 c showsthe metabolic incorporation scheme for a broad spectrum of alkyne-taggedsmall precursors. a.u. arbitrary units. Alkynes possess desirablechemical and spectroscopic features. Chemically, they are small (onlytwo atoms), exogenous (nearly non-existent inside cells), andbioorthogonal (inert to reactions with endogenous biomolecules). Theseproperties render alkynes key players in bioorthogonal chemistry, inwhich precursors labeled with alkyne tags form covalent bonds withazides fused to probes such as fluorophores for detection. However, sucha ‘click-chemistry’ approach prohibits live imaging, as it usuallyinvolves a copper-catalyzed reaction that requires cell fixation, whilethe copper-free version has slow kinetics and high background.Spectroscopically, the C═C stretching motion exhibits a substantialchange of polarizability, displaying a sharp Raman peak around 2125cm⁻¹, which lies in a desirable cell-silent spectral region¹³ (FIG. 10 a). Compared to the popular carbon-deuterium (C-D) Raman tag, alkynesproduce about 40 times higher peaks. However the signal is stillrelatively weak and extremely long acquisition times (˜49 min per frameconsisting of 127×127 pixels) limit dynamic imaging in live systems.

The coupling of SRS microscopy to alkyne tags that we report offerssensitivity, specificity and biocompatibility for probing complex livingsystems. When the energy difference between incident photons from twolasers (pump and Stokes) matches with the 2125 cm-1 mode of alkynevibrations, their joint action will greatly accelerate the vibrationalexcitation of alkyne bonds. As a result of energy exchange between theinput photons and alkynes, the output pump and Stokes beams willexperience intensity loss and gain, respectively. Such intensity changesmeasured by SRS microscopy generate concentration-dependent alkynedistributions in three-dimensions (3D). FIG. 11 is an illustrationshowing a Pump beam (pulsed, pico-second) and an intensity-modulatedStokes beam (pulsed, pico-second) are both temporally and spatiallysynchronized before focused onto cells that have been metabolicallylabeled with alkyne-tagged small molecules of interest. When the energydifference between the Pump photon and the Stokes photon matches thevibrational frequency (Ωvib) of alkyne bonds, alkyne bonds areefficiently driven from their vibrational ground state to theirvibrational excited state, passing through a virtual state. For eachexcited alkyne bond, a photon in the Pump beam is annihilated (Ramanloss) and a photon in the Stokes beam is created (Raman gain). Thedetected pump laser intensity changes through a lock-in amplifiertargeted at the same frequency as the modulation of Stokes beam serve asthe contrast for alkyne distributions.

SRS microscopy offers a number of advantages. First, SRS boostsvibrational excitation by a factor of 107, rendering a quantum leap ofsensitivity (i.e., detectability and speed) over spontaneous Raman.Second, we use a 6-ps pulse width to match the excitation profile ofalkyne (e.g. FIG. 10 a ), assuring efficient and selective nonlinearexcitation. Third, SRS is free of background, whereas spontaneous Ramansuffers from auto-fluorescence and coherent anti-stokes Raman scattering(CARS) suffers from non-resonant background3. Finally, we employnear-infrared laser wavelengths for enhanced tissue penetration,intrinsic 3D sectioning (due to nonlinear excitation) and minimalphoto-toxicity.

We first detected the alkyne-tagged thymidine analogue5-ethynyl-2′-deoxyuridine (EdU) in solution (e.g. FIG. 10 b ). Under afast imaging speed of 100 μs, we determined its detection limit to be200 μM, corresponding to 12,000 alkynes within the laser focus. Thisapproaches the shot-noise limit (ΔIp/Ip˜2×10−7) of the pump beam, whichrepresents the maximum theoretical sensitivity of the system. To explorethe general applicability of our approach, we went on to examine a broadspectrum of small biomolecules including alkyne-taggeddeoxyribonucleoside, ribonucleoside, amino acid, choline and fatty acid(FIG. 10 c ), whose metabolic incorporation has been thoroughly testedin bioorthogonal chemistry studies.

We imaged the metabolic uptake of EdU during de novo DNA synthesis. HeLacells grown in media with EdU show a sharp Raman peak at 2125 cm-1 inthe cell-silent region (e.g. FIG. 12 a ). Live-cell SRS imaging revealedEdU incorporation into the newly synthesized genomes of dividing cells(e.g. FIG. 12 b , alkyne-on). Off-resonance images of the same cells,taken when the energy difference between pump and Stokes photons doesnot match vibrational peaks (alkyne-off), are background-free,confirming the purely chemical contrast of SRS. No EdU signal shows upin cells treated with the DNA synthesis inhibitor hydroxyurea, whereaslipids imaged at 2845 cm-1 verify that these cells are normal based onmorphology. Moreover, we tracked dividing cells every 5 min duringmitosis (e.g. FIG. 12 c ), demonstrating acquisition speed andcompatibility with live dynamics that are nearly impossible withspontaneous Raman. We also showed that our method is applicable tomulticellular organisms. Actively proliferating cells can be clearlydistinguished in C. elegans grown in the presence of EdU as exemplifiedin FIG. 13 , where the composite image shows both the protein derived1655 cm-1 (amide) signal from all the germ cells, and the directvisualization of alkynes (2125 cm-1 (EdU)) highlighting theproliferating germ cells. White circles show examples of EdU positivegerm cells in the mitotic region of C. elegans germline. Scale bar, 5μm.

Next, we studied RNA transcription and turnover using the alkyne-taggeduridine analogue, 5-ethynyl uridine (EU)8 in HeLa cells (e.g. FIG. 12 a). The alkyne-on image (e.g. FIG. 12 d ) reveals localized EU inside thenucleus with higher abundance in the nucleoli, which are majorcompartments of rRNA-rich ribosomal assembly, and nearly disappears inthe presence of the RNA synthesis inhibitor actinomycin D. Turnoverdynamics are further demonstrated by pulse-chase SRS imaging (FIG. 12 e), which indicates a short nuclear RNA lifetime (˜3 h) in live HeLacells.

Many intricate biological processes such as long-term memory requireprotein synthesis in a spatiotemporal dependent manner. We imagedL-Homopropargylglycine (Hpg), an alkyne-tagged analogue of methionine,to visualize newly synthesized proteomes. HeLa cells grown inmethionine-deficient media supplemented with Hpg display an alkyne peak(e.g. FIG. 12 a ) about 20 times lower than that of 10 mM EdU solution(e.g. FIG. 10 a ). The corresponding alkyne-on image (e.g. FIG. 12 f )shows the distribution of newly synthesized proteins with spatialenrichment in the nucleoli (arrow indicated), which experience rapidprotein exchange. Similar to EdU in solution, the detection limit ofalkynes in mammalian cells approaches 200 μM (with 100 μs pixel dwelltime) based on an average signal-to-noise ratio of 2 as we obtained inHeLa cells. The Hpg signal is well retained in fixed cells (e.g. FIG. 14), indicating little contribution from freely diffusing Hpg. Thealkyne-on image displays the Hpg distribution for the newly synthesizedproteins. For the same set of cells, the off-resonant (alkyne-off) imageshows vanishing signal, and the amide image shows total proteindistribution. This result confirms that the detected signal is not fromfreely diffusive precursor Hpg itself (which is eliminated during thefixation process). Scale bar, 10 μm. Furthermore, adding methionine,which has a 500-fold-faster incorporation rate, to compete with Hpgcauses the signal to disappear (e.g. FIG. 12 f ). Note that we verifiedthe spatial patterns of EdU, EU and Hpg incorporation in live cells byperforming click chemistry on fixed cells, with FIGS. 15A-C showing thefluorescence images of HeLa cells incorporated with a, EdU (for DNA); b,EU (for RNA); c, Hpg (for protein). Scale bars, 10 μm.

Lipid metabolism is critical for many functions in healthy and diseasedtissues, but few non-perturbative tags are available to monitor lipidsin the cell. We thus monitored the metabolic incorporation ofalkyne-tagged choline and fatty acids. Hippocampal neurons grown onpropargylcholine present a clear 2142 cm-1 Raman peak (e.g. FIG. 12 a ).Such a frequency shift from 2125 cm-1 is due to the positive charge onthe nitrogen near the alkyne (FIG. 10 c ). As revealed by enzymaticassays (e.g. FIGS. 16 a-16 c ), the alkyne-on signal (FIG. 12 g ) mainlyoriginates from newly synthesized choline phospholipids at membranes. Tolabel fatty acids, we incubated 17-octadecynoic acid (17-ODYA) withTHP-1 macrophages, which actively scavenge cholesterol and fatty acids.In FIG. 16 a , fixed NIH3T3 cells are seen after culturing with 0.5 mMpropargylcholine for 48 hours. The alkyne-on image shows alkyne-taggedcholine distribution. In FIG. 16 b , treatment of fixed NIH3T3 cellswith phospholipase C, which removes Choline head groups of phospholipidsonly in the presence of calcium. The alkyne-on image shows the strongdecrease of incorporated propargylcholine signal, supporting its mainincorporation into membrane phospholipids. For FIG. 16 c , treatment offixed NIH3T3 cells with phospholipase C in the presence of EDTA(chelating calcium). Propargylcholine signal is retained in thealkyne-on image. For FIGS. 16 a-16 c : in the same set of cells as inalkyne-on images, the alkyne-off images show a clear background. Theamide images display total protein distribution. Scale bars, 10 μm. Thealkyne-on image (FIG. 12 h ) depicts the formation of numerous lipiddroplets that indicates transformation into foam cells, a hallmark ofearly atherosclerosis. Multicellular organisms are also capable oftaking up 17-ODYA for lipid imaging. New fatty acids in C. elegansappeared mainly inside lipid droplets upon SRS imaging, known to existlargely in the form of triglycerides (e.g. FIG. 12 i ). Such a fataccumulation process could serve as a useful model for studying obesityand diabetes. We were also able to perform dual-color imaging ofpropargylcholine (2142 cm-1) and EdU (2125 cm-1) incorporation due tothe spectral sharpness and separation of their two alkyne peaks (FIG. 12j ).

Finally, we tracked alkyne-bearing drug delivery (FIGS. 17 a-17 e and 18a-18 b ) in animal tissues by taking advantage of the intrinsic 3Dsectioning property of SRS.

FIG. 17 a depicts Raman spectra of a drug cream, Lamisil, containing 1%TH and mouse ear skin tissue. FIGS. 17 b-17 e illustrate SRS imaging oftissue layers from stratum corneum (z=4 μm) to viable epidermis (z=24μm), sebaceous gland (z=48 μm) and subcutaneous fat (z=88 μm). Tofacilitate tissue penetration, DMSO solution containing 1% TH wasapplied onto the ears of an anesthetized live mouse for 30 min and thedissected ears are imaged afterwards. For all 4 layers shown in FIGS. 17b-17 e , alkyne-on images display TH penetration; alkyne-off images showoff-resonant background (The bright spots in d are due to two-photonabsorption of red blood cells). The composite images show protein (1655cm-1) and lipid (2845 cm-1) distributions. Scale bars, 20 μm.a.u.=arbitrary units.

FIGS. 18 a-18 b show SRS imaging of the viable epidermis layer (z=20 μm)and the sebaceous gland layer (z=40 μm). For both a and b: the alkyne-onimages display the TH penetration into mouse ear tissues through lipidphase. The composite images show both protein (1655 cm-1) and lipid(2845 cm-1) distributions. Scale bars, 20 μm.

Unlike bulky fluorophores, alkynes have little perturbation topharmacokinetics and are common moieties in many pharmaceuticals. Wechose terbinafine hydrochloride (TH), a US Federal Drug Administrationapproved alkyne-bearing antifungal skin drug, and imaged its drugdelivery pathways inside mouse ear tissue to a depth of about 100 μm bytargeting its internal alkyne at 2230 cm-1. TH images captured atvarious depths all exhibit patterns that highly resemble lipiddistributions but not protein distributions, suggesting that THpenetrates into tissues through the lipid phase, consistent with itslipophilic nature. Our technique should be applicable to tracking otherdrugs after proper alkyne derivatization.

In conclusion, we report a general strategy to image small andbiologically vital molecules in live cells by coupling SRS microscopywith alkyne vibrational tags. The major advantages of SRS lie in thesuperior sensitivity, specificity and compatibility with dynamics oflive cells and animals. SRS imaging of alkynes may do for smallbiomolecules what fluorescence imaging of fluorophores has done forlarger species.

Example 3: Synthesis of Bond-Edited Compounds

A. Synthesis of Alkyne-D-Glucose

Sodium hydride (138 mg, 5.8 mmol) was added to a solution of1,2:5,6-Di-O-isopropylidene-α-D-glucofuranose (Compound 51, 500 mg, 1.92mmol, Aldrich D7600) in 10 mL dry DMF at 0° C. The solution was stirredat 0° C. for 30 min before propargyl bromide (80% in toluene, 0.43 mL,3.84 mmol) was added dropwise. The reaction mixture was stirred at roomtemperature for 12 h before quenched with saturated ammonium chloridesolution (10 mL). The mixture was extracted with ethyl acetate (2×25mL), and the organic layer was combined, dried over anhydrous sodiumsulfate, concentrated in vacuo, and purified by column chromatography onsilica gel (0-50% Ethyl acetate in Hexanes) to give Compound S2 (518 mg,90%) as a colorless oil. The 1H NMR spectrum is in accordance withpreviously published values (A. Hausherr et al., Synthesis, 2001, 1377).

1H NMR (400 MHz, CDCl₃) δ 5.88 (d, J=3.6 Hz, 1H), 4.30-4.24 (m, 3H),4.14 (dd, J=7.6, 2.8 Hz, 1H), 4.11-4.06 (m, 2H), 3.99 (dd, J=8.8, 5.6Hz, 1H), 2.47 (t, J=2.4 Hz, 1H), 1.50 (s, 3H), 1.42 (s, 3H), 1.35 (s,3H), 1.31 (s, 3H).

HRMS (FAB+) m/z Calcd. for C₁₅H₂₃O₆ [M+H]⁺: 299.1495. Found: 299.1496.

Water (10 mL) and Dowex® 50WX8 hydrogen form (600 mg, Sigma-Aldrich217514) were added to Compound S2 (594 mg, 1.99 mmol). The mixture washeated to 80° C. for 20 h before filtered. The filtrate was concentratedin vacuo to give Compound S3 (416 mg, 1.91 mmol, 96%) as a white solid.

1H NMR (400 MHz, D₂O) δ 5.13 (d, J=3.6 Hz, 1H), 4.44 (d, J=2.4 Hz, 2H),3.79-3.73 (m, 2H), 3.70-3.61 (m, 2H), 3.51 (dd, J=9.8, 3.8 Hz 1H), 3.40(t, J=9.6 Hz, 1H), 2.82 (s, 1H). 13C NMR (101 MHz, D₂O) δ 92.1, 80.8,79.8, 75.9, 71.4, 71.2, 69.2, 60.4, 59.9.

HRMS (FAB+) m/z Calcd. for C9H14O6Na [M+Na]+: 241.0688. Found: 241.0683.

B. Synthesis of EdU-¹³C (Compound 2) and EdU-¹³C₂ (Compound 3)

Synthesis of Compound 4

To a solution of 5-iodo-2′-deoxyuridine (Compound 1, 150 mg, 0.42 mmol)in 1.5 ml of pyridine was added 0.4 ml (0.42 mmol) acetic anhydride at0° C. The resulting mixture was warmed up to room temperature andstirred for 4 h, then poured into 5 ml of cold 1 N NaHSO₄ and extractedwith ethyl acetate three times. The organic layer was washed withsaturated NaHCO₃ and brine, dried over anhydrous Na₂SO₄ andconcentrated. The crude product was purified by column chromatography onsilica gel (0-70% Ethyl acetate in Hexanes) to give Compound 4 (157.3mg, 0.36 mmol, 85%) as a white solid.

1H NMR (400 MHz, CDCl₃) δ ppm: 8.46 (s, 1H), 7.97 (s, 1H), 6.28 (dd,J=8.2, 5.7 Hz, 1H), 5.27-5.19 (m, 1H), 4.41 (dd, J=12.3, 3.2 Hz, 1H),4.34 (dd, J=12.3, 2.9 Hz, 1H), 4.30 (q, J=2.9 Hz, 1H), 2.54 (ddd,J=14.3, 5.7, 2.1 Hz, 1H), 2.21 (s, 3H), 2.20-2.13 (m, 1H), 2.12 (s, 3H).

MS (APCI+) m/z Calcd. for C₁₃H₁₆IN₂O₇ [M+H]⁺: 439.0. Found: 438.8.

Synthesis of Compound 5

To an oven-dried vial was added Compound 4 (72 mg, 164 μmol), Pd (OAc)2(3.6 mg, 16 μmol), PPh3 (8.6 mg, 33 μmol), Cut (3.1 mg, 16 μmol), DMF (2ml), Et3N (50 mg, 69 492 μmol) and TMS13C≡13CH (25 mg, 250 μmol) underAr. The yellow mixture was stirred at RT for 15 h before concentrated invacuo. The residue was purified by column chromatography on silica gel(0-70% Ethyl acetate in Hexanes) to give Compound 5 (48.4 mg, 118 μmol,72%) as a thin film.

1H NMR (400 MHz, Methanol-d4) δ 7.98 (d, J=5.0 Hz, 1H), 6.23 (dd, J=7.8,6.0 Hz, 1H), 5.28 (dt, J=6.7, 2.6 Hz, 1H), 4.36 (t, J=3.1 Hz, 2H),4.34-4.28 (m, 1H), 2.50 (ddd, J=14.5, 6.0, 2.5 Hz, 1H), 2.39 (ddd,J=14.5, 7.9, 6.6 Hz, 1H), 2.16 (s, 3H), 2.09 (s, 3H), 0.20 (d, J=2.5 Hz,9H). 13C NMR (101 MHz, MeOD) δ 99.54 (d, J=140.5 Hz), 96.95 (d, J=140.5Hz).

MS (APCI+) m/z Calcd. for C1613C2H25N2O7Si [M+H]+: 411.2. Found: 411.0Synthesis of Compound 3:

To a solution of Compound 5 (3.5 mg, 8.5 μmol) in 0.9 ml MeOH and 0.1 mlH₂O was added K2CO3 (6.0 mg, 43 μmol) at RT. The reaction was stirredovernight before concentrated in vacuo. The residue was purified byreverse phase HPLC to give compound 3 (1.6 mg, 6.4 μmol, 75%) as a thinfilm.

HPLC condition: 20 min gradient elution using H2O:MeCN starting from100:0 to 85:15. Retention time: 15.4 min

1H NMR (400 MHz, MeOD) δ: 8.39 (d, J=5.6 Hz, 1H); 6.24 (t, J=6.4 Hz,1H); 4.40 (m, 1H); 3.94 (dd, J=6.4, 3.2 Hz, 1H); 3.82 (dd, J=12, 3.2 Hz,1H); 3.73 (dd, J=12, 3.6 Hz, 1H); 3.53 (dd, J=250.4, 54.8 Hz, 1H); 2.32(ddd, J=13.6, 6, 3.6 Hz, 1H); 2.23 (m, 1H). 13C NMR (101 MHz, MeOD) δ82.87 (d, J=180.4 Hz), 75.85 (d, J=180.3 Hz).

MS (FAB+) m/z Calcd. for C913C2H13N2O5 [M+H]⁺: 255.09. Found: 255.11.

Synthesis of Compound 9

To a solution of ethynylmagnesium bromide in THF (5.0 ml, 0.5 Msolution, 2.5 mmol) was added 15 ml THF under Ar. The solution wascooled to −78° C. and 2.4 ml n-BuLi in hexane (1.6 M, 3.8 mmol) wasadded dropwisely. After 30 min, chloro(dimethyl)octylsilane (1.21 ml,1.06 g, 5.1 mmol) was added dropwisely. The reaction was then warmed toRT and stirred for another 3h before filtered through a short pad ofsilica. The solvent was removed under reduced pressure and the residuewas purified by column chromatography on silica gel (pure Hexanes) togive Compound 9 (885 mg, 2.4 mmol, 96%) as a colorless liquid.

1H NMR (400 MHz, Chloroform-d) δ 1.42-1.24 (m, 24H), 0.88 (t, J=6.6 Hz,6H), 0.60 (dd, J=9.4, 6.2 Hz, 4H), 0.13 (s, 12H). 13C NMR (101 MHz,Chloroform-d) δ 113.94, 33.37, 32.12, 29.49, 29.43, 23.92, 22.85, 16.26,14.27, −1.55.

HRMS (EI+) m/z Calcd. for C₂₂H₄₆Si₂ [M]⁺:366.3138. Found: 366.3134.

Synthesis of Compound 10

In a glove box filled with Ar, catalyst 8 (36.5 μma 5 eq.) was preparedin 0.5 mL dry CCl4 in situ according to the procedure documented byJyothish and Zhang (Angew. Chem. Int. Ed. Engl. 50, 3435-8 (2011)). Tothe solution of catalyst 8 in CCl4 was added 9 (267 mg, 0.73 mmol) and asolution of Compound 5 (3.0 mg, 7.3 μmol) in 0.5 mL dry CCl4. Themixture was heated to 70° C. for 8 h before concentrated in vacuo. Theresidue was purified by column chromatography on silica gel (0-70% Ethylacetate in Hexanes) to recover Compound 5 (0.5 mg, 1.2 μmol) and to giveCompound 10 (1.0 mg, 2.0 μma 27%, 33% B.R.S.M.) as a thin film.

1H NMR (400 MHz, Methanol-d4) δ 7.97 (d, J=5.6 Hz, 1H), 6.23 (dd, J=7.9,5.9 Hz, 1H), 5.28 (dt, J=6.8, 2.4 Hz, 1H), 4.36 (dd, J=5.8, 3.4 Hz, 2H),4.31 (dd, J=6.3, 3.2 Hz, 1H), 2.50 (ddd, J=14.5, 6.1, 2.5 Hz, 1H), 2.38(ddd, J=20.2, 7.7, 6.1 Hz, 1H), 2.15 (s, 3H), 2.09 (s, 3H), 1.30 (s,12H), 0.90 (t, J=6.9 Hz, 3H), 0.70-0.62 (m, 2H), 0.18 (s, 6H). 13C NMR(101 MHz, MeOD) δ 97.56.

MS (FAB+) m/z Calcd. for C2413CH38N2NaO7Si [M+Na]+: 530.24. Found:530.25.

Synthesis of Compound 2

To a solution of compound 10 (0.4 mg, 0.8 μmol) in 0.5 ml MeOH and 0.05ml H2O was added K2CO3 (2.0 mg, 14 μmol) and TBAF (20 μL, 1 M in THF) atRT. The reaction was stirred 7 h at RT before concentrated in vacuo. Theresidue was purified by reverse phase HPLC to give compound 2 (0.1 mg,0.4 μmol, ˜50%) as a thin film.

HPLC condition: 20 min gradient elution using H2O:MeCN starting from100:0 to 85:15. Retention time: 15.4 min

The mass of the product is determined by UV-Vis (λabs=288 nm, ε=12,000cm-1M-1 in methanol).

1H NMR (500 MHz, Methanol-d4) δ 8.39 (d, J=5.7 Hz, 1H), 6.24 (t, J=6.5Hz, 1H), 4.40 (dt, J=6.6, 3.6 Hz, 1H), 3.94 (q, J=3.3 Hz, 1H), 3.82 (dd,J=12.0, 3.1 Hz, 1H), 3.73 (dd, J=12.0, 3.4 Hz, 1H), 3.53 (d, J=51.3 Hz,1H), 2.32 (ddd, J=13.6, 6.2, 3.7 Hz, 1H), 2.27-2.17 (m, 1H). 13C NMR(101 MHz, MeOD) δ 76.00. MS (ESI+) m/z Calcd. for C1013CH13N2O5 [M+H]+:254.09. Found: 254.70.

Synthesis of EU-¹³C₂ (Compound 13)

Synthesis of S6:

To an oven-dried vial was added compound S5 (15 mg, 50 μmol), Pd(OAc)₂(1.1 mg, 5 μmol), PPh3 (2.6 mg, 10 μmop CuI (1.0 mg, 5 μmol), DMF (1ml), Et3N (15 mg, 20.7 μl, 150 μmol) and TMS13C13CH (7.5 mg, 10.8 μl, 75μmol) under Ar. The mixture was stirred at RT for 12 h beforeconcentrated in vacuo. The residue was purified by column chromatographyon silica gel (0-50% methanol in dichloromethane) to give compound S6(9.0 mg, 26 μmol, 52%) as a thin film.

1H NMR (400 MHz, Methanol-d4) δ 8.41 (d, J=4.9 Hz, 1H), 5.91-5.83 (m,1H), 4.21-4.13 (m, 2H), 4.07-3.98 (m, 1H), 3.88 (dd, J=12.2, 2.6 Hz,1H), 3.75 (dd, J=12.2, 2.8 Hz, 1H), 0.20 (d, J=2.3 Hz, 9H). 13C NMR (101MHz, MeOD) δ 99.24 (d, J=141.0 Hz), 96.95 (d, J=141.0 Hz).

MS (FAB+) m/z Calcd. for C1213C2H21N2O6Si [M+H]+: 343.12. Found: 343.17.

Synthesis of Compound 13

To a solution of compound S6 (3.0 mg, 8.8 μmop in 0.6 ml MeOH and 0.1 mlH2O was added K2CO3 (5.0 mg, 36 μmop at RT. The reaction was stirredovernight before concentrated in vacuo. The residue was purified byreverse phase HPLC to give compound 13 (2.2 mg, 8.1 μmol, 92%) as a thinfilm.

1H NMR (400 MHz, Methanol-d4) δ 8.47 (d, J=5.6 Hz, 1H), 5.93-5.83 (m,1H), 4.21-4.13 (m, 2H), 4.06-3.98 (m, 1H), 3.88 (dd, J=12.2, 2.6 Hz,1H), 3.75 (dd, J=12.2, 2.8 Hz, 1H), 3.54 (dd, J=250.4, 54.6 Hz). 13C NMR(101 MHz, MeOD) δ 82.90 (d, J=180.2 Hz), 75.74 (d, J=180.2 Hz).

MS (ESI+) m/z Calcd. for C913C2H13N206 [M+H]+: 271.08. Found: 271.51.

FIG. 38 shows a block diagram of an exemplary embodiment of a systemaccording to the present disclosure. For example, exemplary proceduresin accordance with the present disclosure described herein can beperformed by a processing arrangement and/or a computing arrangement3802. Such processing/computing arrangement 3802 can be, for exampleentirely or a part of, or include, but not limited to, acomputer/processor 3804 that can include, for example one or moremicroprocessors, and use instructions stored on a computer-accessiblemedium (e.g., RAM, ROM, hard drive, or other storage device).

As shown in FIG. 38 , for example a computer-accessible medium 3806(e.g., as described herein above, a storage device such as a hard disk,floppy disk, memory stick, CD-ROM, RAM,

ROM, etc., or a collection thereof) can be provided (e.g., incommunication with the processing arrangement 3802). Thecomputer-accessible medium 3806 can contain executable instructions 3808thereon. In addition or alternatively, a storage arrangement 3810 can beprovided separately from the computer-accessible medium 3806, which canprovide the instructions to the processing arrangement 3802 so as toconfigure the processing arrangement to execute certain exemplaryprocedures, processes and methods, as described herein above, forexample.

Further, the exemplary processing arrangement 3802 can be provided withor include an input/output arrangement 3814, which can include, forexample a wired network, a wireless network, the internet, an intranet,a data collection probe, a sensor, etc. As shown in FIG. 38 , theexemplary processing arrangement 3802 can be in communication with anexemplary display arrangement 3812, which, according to certainexemplary embodiments of the present disclosure, can be a touch-screenconfigured for inputting information to the processing arrangement inaddition to outputting information from the processing arrangement, forexample. Further, the exemplary display 3812 and/or a storagearrangement 3810 can be used to display and/or store data in auser-accessible format and/or user-readable format.

Exemplary Imaging Intracellular Fluorophores with Sub-MicromolarSensitivity Using Pre-Resonance Stimulated Raman Scattering

The exemplary system, method and computer-accessible medium, accordingto an exemplary embodiment of the present disclosure, can utilize pr-SRSmicroscopy and apply such exemplary procedure to image molecules in thepre-resonance Raman regime to achieve both superb sensitivity atsub-micromolar concentration, and chemical specificity for multipleximaging (see, e.g., FIG. 39 a ). SRS microscopy can be a nonlineartwo-laser pump-probe Raman technique, utilizing the stimulated emissionquantum amplification from the Stokes beam photons in addition to pumpbeam photons to facilitate the intrinsically weak Raman transition.(See, e.g., References 99 and 100). The effective stimulated Raman crosssection (σ_(st)) can be defined compared to a spontaneous Raman crosssection (σ_(sp)) as, for example:

σ_(st)=σ_(sp) G(p _(w) _(s) )  (1a)

where G(p_(w) _(s) ) can be the stimulated Raman gain factor dependingon the Stokes beam power (p_(w) _(s) ). The measured G(p_(w) _(s) ) canbe about 107-108 with a biologically compatible Stoke beam power. (See,e.g., Reference 100). With such powerful quantum amplification, SRSmicroscopy has been applied in a great deal of biological studies suchas video-rate imaging in vivo, brain tumors detection and drug deliverytracking. (See, e.g., References 101-103). In addition, as a nonlinearmicroscopy that can utilize near-infrared excitation wavelength, SRSalso offers both subcellular resolution and intrinsic 3D sectioning fordeep tissue imaging.

However, current SRS sensitivity can still be far from that offluorescence microscopy. The measured SRS detection limit of a typicalchemical bond such as carbon-hydrogen bond (C—H, σ_(sp)˜10⁻³⁰ cm²) canbe about 15 mM. (See, e.g., Reference 99). Even for chemical bonds withexceptional strong Raman polarizability, such as alkynes (C≡C), thereported detection limit can be about 200 μM. (See, e.g., Reference103). Efforts have been devoted to synthesize molecules with strongerRaman vibration, however, only a few molecules present such property,and the improvements can typically only be about 27 times. (See, e.g.,Reference 104). Presently, all the SRS applications focus on probingmolecules in the non-resonance region where the absorption peak energy(ω0) of the molecules can be much larger than the pump laser energy(ωpump). (See, e.g., FIG. 39 b ). One way to further improve SRSsensitivity can be by moving from non-resonance Raman to resonanceRaman, where ω0−ωpump<Γ (e.g., Γ can be the homogeneous line width for amolecule, typically, Γ˜800 cm-1), thus achieving Raman intensity gainbenefiting from the coupling of electronic transition. In this exemplarycase, the resonance enhanced spontaneous Raman cross-section σ_(sp,RE)could be shown as, for example:

σ_(sp,RE)=σ_(sp) G _(RE)  (2a)

where, G_(RE) can be the resonance enhanced Raman gain factor, which canincrease when the energy difference between ω0 and ωpump can decrease.Furthermore, the effective resonance enhanced SRS cross-section can beexpressed as, for example:

σ_(st,RE)=σ_(sp) G _(RE) G(p _(w) _(s) )  (3a)

To illustrate the resonance Raman enhancement in the nonlinear SRSmicroscopy, for example, a non-resonance SRS spectrum of Coumarin 153can be measured, which can show an absorption peak wavelength (λmax) atabout 422 nm (e.g., 23697 cm-1, ω0>>ωpump) (see, e.g., FIG. 39 b ) and arigorous resonance SRS spectrum of IR895 with λmax=895 nm (e.g., about11173 cm-1, ω0−ωpump=0) (see, e.g., FIG. 39 c ) by sweeping the pumplaser wavelength across the range of about 895-940 nm (e.g., 11173-10638cm-1) with a fixed Stokes laser wavelength at about 1064.2 nm (e.g.,9398 cm-1). The measured Raman cross-section for the conjugatedcarbon-carbon double bond (C═C) for IR895 (see, e.g., FIG. 39 c ,element 3905) can approach about 104 as compared to that of Coumarin153, while C═C of Coumarin 153 may only be about 1.5 times of C≡C (e.g.,FIG. 39 b , element 3910). However, with the increasing of the Ramanintensity, a large background can appear possibly due to the competingpump-probe process of simultaneous excitation saturation, and stimulatedemission of the electronic transition for IR895. Moreover, the measuredRaman peak for IR895 can present an inverse Lorentzian shape due to therigorous resonance Raman measurement for the stimulated Raman losssignal. (See, e.g., Reference 105). For a spectroscopy measurement,these complications may not be an issue with post-acquisitionprocessing. For example, femtosecond SRS spectra of rigorous resonantfluorophores have been heavily documented and the appearance of such anintense background is also well known. (See, e.g., References 106 and107). Nevertheless, such a background can be extremely undesirable forimaging applications, which can benefit from direct visualization ofspatial information.

Close inspection for both the non-resonance SRS spectrum of Coumarin 153(see, e.g., FIG. 39 b ) and rigorous resonance SRS spectrum of IR895(see, e.g., FIG. 39 c ) can indicate that a rigorous resonance can bringbackground more than vibrational signals. Therefore, an exemplarydetuning from rigorous resonance can reduce the electronically relatedbackground faster than the vibrational signal. For example, this can berationalized since the electronically related background can benefitfrom the excitation of the real population, which can vanish if the pumplaser energy does not match with the excitation energy, while thevibrational signal can be mediated by virtual state. Indeed, thisrationalization can be supported by the SRS spectrum of Cy7.5, which canshow a ratio of approximately 0.3 between on-resonance Raman signal andoff-resonance Raman signal with a detuning of ω0−ωpump˜2Γ (see, e.g.,Figure. 39c) compared to that of 0.03 for IR895 when ω0−ωpump˜0. (See,e.g., FIG. 39 b ). A further detuning to ˜3Γ pinpointed an ATTO740 dye(λmax=740 nm) with both a Raman intensity gain of 1000 times compared toC≡C and a clean off-resonance background (see, e.g., FIG. 39 d ) can beperformed. This can be the so-called pre-resonance Raman region, whichhas been revealed, and can be the most suitable regime for SRS imagingfor both high sensitivity and chemical specificity. This can be calledthe pr-SRS region when ω0−ωpump can fall within the range of 3Γ-5Γ.

An exemplary configuration and principle for pre-resonance SRSmicroscopy according to an exemplary embodiment of the presentdisclosure, is shown in FIGS. 39 a-39 e . For example, FIG. 39 aillustrates a diagram of an exemplary SRS system. FIG. 39 b illustratesan exemplary energy diagram and the corresponding non-resonance SRSspectrum of 4 mM Coumarin 153 in DMSO under power of Ppump=24 mW,Pstokes=24 mW. FIG. 39 c shows an exemplary energy diagram and thecorresponding rigorous resonance SRS spectrum of 10 μM IR895 in methanolunder power of Ppump=1.2 mW, Pstokes=9.6 mW. FIG. 39 d provides anexemplary energy diagram and the corresponding resonance SRS spectrum of100 μM Cy7.5 in DMSO under power of Ppump=12 mW, Pstokes=9.6 mW. FIG. 39e shows an exemplary energy diagram and the corresponding pre-resonanceSRS spectrum of 100 μM ATTO740 in DMSO under power of Ppump=24 mW,Pstokes=24 mW. For example, elements 3905, 3910, 3915 and 3920 canindicate the highest Raman peaks in the molecules that attribute to theC═C bond vibration.

To illustrate further the pr-SRS enhancement between the experimentresults and theory, FIG. 40 a shows measured σ_(sp)(C═C) for 25different fluorophores over a wide range of ω0. Assuming the molecularstructure variation can be negligible in the consideration forpre-resonance Raman enhancement factor, the graph of FIG. 40 a wasover-plotted with the theoretically approximated pr-Raman cross-section30 over the same ω0 range of, for example:

$\begin{matrix}{\sigma_{RE} = {K{\omega_{0}\left( {\omega_{0} - \omega_{vib}} \right)}^{3}\left( \frac{\left( {\omega_{pump}^{2} + \omega_{0}^{2}} \right)}{\left( {\omega_{0}^{2} - \omega_{pump}^{2}} \right)^{2}} \right)^{2}}} & \left( {4a} \right)\end{matrix}$

where ωvib can be the vibrational transition energy. Based on the above,it was determined that the pre-resonance enhancement from theoreticalcalculations can match very well with the experimental results. The greyshaded area 4005 from FIG. 40 a indicates the selected fluorophores mostsuitable for pr-SRS imaging with both high enough pre-resonantenhancement and chemical specificity. For the dyes in the shaded area4005 from FIG. 40 a , ATTO740 can be the best dye presenting apre-resonance enhancement gain (G_(pRE)) of 105 when compared withσ_(sp)(C—H) thus σ_(st,pRE)(ATTO740) can reach 10-18 cm⁻² with anexperimental demonstrated G(p_(w) _(s) )˜108, approaching that of singlemolecule absorption cross-section at 10-16 cm′.

With such sensitivity, both high sensitivity and chemical specificityfor the exemplary pr-SRS technique in solutions can be analyzed. Byappropriately selecting the imaging condition with excitation power thatcan be low enough not to damage the fluorophore but high enough tomildly saturate the Raman transition of ATTO740, the measured detectionlimit with shot-noise limited sensitivity for ATTO740 can be about 0.7μM with about a 1 ms time constant and about a 2 μM with about a 100 μstime constant that can be suitable for live-cell imaging (see, e.g.,FIG. 40 b ). For pr-SRS, which can still probe the virtual-statemediated Raman transition benefiting from pre-resonance enhancement, thepump laser may not directly excite the fluorophore to the realelectronic state. Therefore the photobleaching damage can be minimum. Todemonstrate the high chemical specificity in pr-SRS, 3 dyes (Cy5,sulfo-Cy5, ATTO655) that absorb similarly around 650 nm can be selected.While the three dyes (e.g., Cy5 (e.g., element 4025), Sulfo-Cy5 (e.g.,element 4030) and ATTO 655 (e.g., element 4035)) present almostindistinguishable absorption and emission spectra (see, e.g., FIG. 40 c), ATTO655, as an oxazine dye, shows a C═C Raman peak at 1664 cm⁻¹ (see,e.g., FIG. 40 d element 4010) that can be completely distinguishablefrom the C═C peak of cyanine dyes (Cy5, sulfo-Cy5) at 1606 cm⁻¹ (e.g.,FIG. 40 d , element 4010). In addition, sulfo-Cy5 can also bedistinguishable from Cy5 between the 1372 cm⁻¹ peak and the 1359 cm⁻¹peak (see, e.g., FIG. 40 d , elements 4015 and 4020) even with a minorchemical modification of aromatic sulfonation. Thus, it can be shownspectroscopically that fluorophores with inseparable electronicspectroscopy can be easily differentiated in Raman spectrum because ofthe chemical selectivity from vibrational spectroscopy.

FIGS. 40 a-40 d show exemplary graphs of an exemplary pr-SRS signal withsuperb sensitivity and chemical specificity. For example, FIG. 40 aillustrates a semi-log plot of the measured Raman cross-section for 25fluorophores with various absorption peak energies. Grey shaded-area4005 of FIG. 40 a indicates the selected pre-resonance SRS region. FIG.40 b illustrates the linear dependence of stimulated Raman loss signalsat 1642 cm⁻¹ with ATTO740 concentrations under an about 100 μs timeconstant. FIG. 40 c illustrates the overlapping absorption and emissionspectra of Cy5, sulfo-Cy5 and ATTO655. FIG. 40 d illustrates the SRSspectra of Cy5, sulfo-Cy5 and ATTO655 with discernible Raman peaks.

Pr-SRS imaging on intracellular fluorophores can be provided which haveachieved exceptional image contrast with a panel of immuno-labeledspecific types of intracellular proteins including tubulin, Tom20 (e.g.,mitochondria marker), giantin (e.g., Golgi marker) and neurofilamentheavy proteins (e.g., Neuronal Marker) with ATTO740 and Dylight650 dyesin either cultured hippocampal neurons or HeLa cells. (See, e.g., FIGS.41 a-41 e ). Such high quality SRS images can be obtained for anindividual type of proteins labeled with fluorophores. Besides theheightened imaging sensitivity, the specific chemical selectivity usingthe exemplary system, method, and computer-accessible medium accordingto an exemplary embodiment of the present disclosure can also beattained, where the on-resonant image at 1642 cm⁻¹ (λpump=905.9 nm) forATTO740-labeled tubulin can be clearly shown, but the correspondingoff-resonance signal at 1702 cm-1 (λpump=901 nm) can vanish when theenergy difference between the pump and Stokes photons does not match theωvib for the C═C vibrational transition of ATTO740. Such importantchemical specificity can extend beyond an achievement for standardfluorescence-based detection systems as illustrated in FIGS. 41 g-41 j ,in which the Alexa647 labeled 5-Ethynyl-2′-deoxyuridine (“EdU”) for DNAdetection shows a clear Raman vibrational on-off resonance contrast bytuning λpump just to about 2 nm away from the peak. (See, e.g., FIGS. 41g and 41 h ). Further, the two-photon fluorescence signal remains thesame by tuning the excitation laser wavelength by about 2 nm off thepeak. (See, e.g., FIGS. 41 i and 3 j ).

Indeed, FIGS. 41 a-41 j show exemplary pr-SRS images with superbsensitivity and chemical specificity. For example, FIG. 41 a illustratesan ATTO740 immuno-labeled tubulin in hippocampal neurons targeting the1642 cm-1 peak. FIG. 41 b illustrates an ATTO740 immuno-labeled tom20 inHeLa cells at 1642 cm-1. FIG. 41 c illustrates an ATTO740 immuno-labeledgaintin in HeLa cells at 1642 cm-1. FIG. 41 d illustrates a Dylight650immuno-labeled neurofilament heavy protein in hippocampal neurons at1606 cm-1. FIG. 41 e illustrates an on-resonance ATTO740 immuno-labeledtubulin in HeLa cells at 1642 cm-1. FIG. 41 f illustrates anoff-resonance signal at 1702 cm-1 on the same HeLa cells as in FIG. 41 e. FIG. 41 g illustrates an on-resonance Alexa647 labeled5-Ethynyl-2′-deoxyuridine for newly synthesized DNA in HeLa cells at1606 cm-1. FIG. 41 h illustrates an off-resonance image at 1580 cm-1 onthe same HeLa cells as in FIG. 41 g . FIGS. 41 i and 41 j illustratetwo-photon fluorescence images of the same HeLa cells as in FIG. 41 g atabout 810 nm (see e.g., FIG. 41 i ) and about 812 nm (see e.g., FIG. 41j ) of the two-photon excitation peak of Alexa647.

Thus, from both the spectroscopy and imaging perspectives, the superbsensitivity and the distinct chemical specificity for the exemplarypr-SRS of fluorophores is shown. With such sensitivity and specificity,the uses of the exemplary system, method and computer-accessible mediumfor biomedical researches, among which large number multi-color imagingcan be important. For example, FIG. 42 a illustrates an exemplary graphof a 6-color channel pr-SRS multiplex imaging possibility with 6different fluorophores which include 80 μm Atto740 (e.g., element 4205),100 μm Atto 700 (e.g., element 4210), 250 μm Cy5.5 (e.g., element 4215),500 μm Alexa647 (e.g., element 4220), 200 μm Atto665 (e.g., element4225) and 500 μm Dylight650 (e.g., element 4230). Among the differentfluorophores shown, each of the three dyes shows an almost overlappingabsorption and emission spectra. By specifically selecting the target,FIG. 42 b demonstrates the 4-color pr-SRS imaging for intracellularfluorophores with distinct and quantitative signal separation of Cy5.5(λmax=673 nm) labeled newly synthesized DNA, ATTO740 (λmax=740 nm)labeled nucleoli fibrillarin proteins, Atto700 (λmax=700 nm) labeleda-tubulin and Alexa647 labeled newly synthesized proteins after linearcombinational procedure. The 4-color overlay image (see, e.g., FIG. 42 c) presents clearly defined spatial relationships between the targetedfour types of molecules. Moreover, since SRS signal can be orthogonalwith fluorescence signal, besides the multiplex pr-SRS imaging alone,tandem imaging with the fluorescence could further expand the numbers ofsimultaneously detected colors. To demonstrate such configuration, aDAPI (λmax=350 nm) that stains DNA, dylight488 (λmax=488 nm) stainingcalnexin (e.g., ER marker) and mito-tracker orange (e.g., 543 nm)staining mitochondria for three-color fluorescence imaging, in additionto the above 4-color multiplex pr-SRS imaging in the same set of cells,can be selected (see, e.g., exemplary images of FIG. 42 d ). Since, forexample, the 4 dyes for pr-SRS imaging can all be with an absorptionpeak at about 650-740 nm with far-red emission, and the 3 dyes forfluorescence imaging can be around 350-543 nm that can be in thenon-resonance SRS region with minimum resonance enhancement, both thefluorescence signal and the pr-SRS signal can be free of interferencefrom each other. Thus, the exemplary system, method andcomputer-accessible medium can achieve at least 7-color imaging.

In particular, FIGS. 42 a-42 d illustrate simultaneous 7-colorquantitative pr-SRS and fluorescence tandem imaging. For example, FIG.42 a illustrates pr-SRS spectra with 6 possible colors for 6fluorophores of ATTO740 (1642 cm-1), ATTO700 (1657 cm-1), Cy5.5 (1626cm-1), alexa647 (1606 cm-1, 1358 cm-1), ATTO655 (1665 cm-1) andDylight650 (1606 cm-1, 1358 cm-1). FIG. 42 b illustrates a 4-colorquantitative pr-SRS imaging of Cy5.5 labeled EdU for newly synthesizedDNA; ATTO740 immuno-labeled Fibrillarin protein of nucleolar marker;ATTO740 immuno-labeled alpha-tubulin; Alexa657 labeled AHA for newlysynthesized proteins in the same HeLa cells. FIG. 42 c illustrates anoverlay of 4-color pr-SRS images in FIG. 42 b . FIG. 42 d illustratestandem fluorescence imaging of DAPI labeled DNA; Dylight488immuno-labeled Calnexin protein of Endoplasmic reticulum marker;Mitotracker orange labeled mitochondria.

Additional exemplary labels can be created for pr-SRS palette usingvarious other vibrational moieties. Unlike C═C, which can exhibitmultiple peaks in the crowded fingerprint region, triple bonds,including alkyne or nitrile, can display a single sharp Raman peak inthe wide silent window (e.g., from about 1800 to about 2800 cm-1) freefrom cellular background. Thus pr-SRS imaging of triple bonds cangreatly expand a vibrational palette with minimum cross talks. This canbe non-trivial because triple bonds may need to be coupled with anelectronic transition in order to gain resonance enhancement. Thus,described herein is a new family of vibrational dyes in which triplebonds can directly participate in the π-conjugation systems. Forexample, general dye scaffolds with optimal conjugation position oftriple bonds were determined, and then their absorption peaks were tunedinto the pr-SRS region, ensuring both intensity and chemicalspecificity. To generate more vibrational colors, an isotopic edition onthe triple bonds was utilized in conjunction with exquisiteelectron-density tuning on the π-conjugation system to shift the peakfrequency. The resulting 10 exemplary reporters, termed Manhattan Ramanscattering (“MARS”) dyes (see e.g., FIGS. 43 a-43 d : e.g., MARS2242element 4305, MARS2228 element 4310, MARS2215 element 4315, MARS2200element 4320, MARS2186 element 4325, MARS2176 element 4330, MARS2159element 4335, MARS2148 element 4340, MARS2114 element 4345 and MARS2061element 4350), display well-resolved individual pr-SRS peaks in acell-silent window. Except for MARS2124 whose pr-SRS spectrum canexhibit some background likely from two-photon absorption, the other 9exemplary MARS dyes have negligible (e.g., about <15% on average)spectral cross-talk under the narrow-band (e.g., about 6 cm-1) laserexcitation.

Additionally, the exemplary system, method and computer-accessiblemedium, according to an exemplary embodiment of the present disclosure,can utilize pr-SRS that can achieve an increased detection sensitivitydown to sub-micromole and high chemical specificity for multicolorimaging. For example, a narrow region of the absorption peaks of dyescan be selected that can be suitable for pr-SRS imaging, which canbenefit from pre-resonance Raman enhancement, but does not suffer fromany other competing pump-probe signal contributing to a largeoff-resonance background. With such sensitivity and specificity, 4-colorimaging can be achieved by commercially available dyes with pr-SRSalone, which can already be comparable to the typical number limit inmulti-color fluorescence imaging. In tandem with fluorescencemicroscopy, simultaneous imaging of 3 more colors can be obtained, thus,almost doubling the number limit of multicolor fluorescence imaging.This number can be expanded furthermore with custom synthesizedmolecules leading to more resolvable pr-SRS colors.

Exemplary SI Methods and Materials

An integrated laser (e.g., picoEMERALD with custom modification, AppliedPhysics & Electronics, Inc.) can be used as the light source for bothpump and Stokes beams. picoEMERALD can provide an output pulse train at1064 nm with 6 ps pulse width and 80 MHz repetition rate, which servesas the Stokes beam. The frequency-doubled beam at 532 nm can be used tosynchronously seed a picosecond optical parametric oscillator (“OPO”) toproduce a mode-locked pulse train (e.g., the idler beam of the OPO canbe blocked with an interferometric filter) with 5-6 ps pulse width. Thewavelength of the OPO can be tunable from about 720 to about 990 nm,which can serve as the pump beam. The intensity of the about 1064 nmStokes beam can be modulated sinusoidally by a built-in electro-opticmodulator (“EOM”) at about 8 MHz with a modulation depth of more thanabout 95%. The pump beam can be spatially overlapped with the Stokesbeam with a dichroic mirror inside picoEMERALD. The temporal overlapbetween pump and Stokes pulse trains can be ensured with a built-indelay stage and optimized by the SRS signal of pure dodecane liquid.

Pump and Stokes beams can be coupled into an inverted laser-scanningmicroscope (e.g., FV1200MPE, Olympus) optimized for near IR throughput.An about 60× water objective (UPlanAPO/IR, 1.2 N.A., Olympus) with highnear IR transmission can be used for all cellular level imaging, and a25× water objective (XLPlan N, 1.05 N.A., MP, Olympus) with both highnear IR transmission and a large field of view can be used for braintissue and in vivo imaging. The Pump/Stokes beam size can be matched tofill the back-aperture of the objective. The forward going Pump andStokes beams, after passing through the sample, can be collected intransmission with a high N.A. condenser lens (e.g., oil immersion, 1.4N.A., Olympus), which can be aligned following Kohler illumination. Atelescope can then be used to image the scanning mirrors onto a largearea (e.g., about 10 mm by about 10 mm) Si photodiode (e.g., FDS1010,Thorlabs) to descan beam motion during laser scanning. The photodiodecan be reverse-biased by about 64 V from a DC power supply to increaseboth the saturation threshold and response bandwidth.

A high optical density (“O.D.”) bandpass filter (e.g., 890/220 CARS,Chroma Technology) can be used to block the Stokes beam completely, andtransmit the Pump beam only. The output current of the photodiode can beelectronically pre-filtered by an about 8-MHz band-pass filter (e.g., KR2724, KR electronics) to suppress both the 80 MHz laser pulsing and thelow-frequency contribution due to laser scanning across the scatteringsample. It can then be fed into a radio frequency lock-in amplifier(e.g., SR844, Stanford Research Systems) terminated with about 50Ω todemodulate the stimulated Raman loss signal experienced by the pumpbeam. The in-phase X-output of the lock-in amplifier can be fed backinto the analog interface box (e.g., FV10-ANALOG) of the microscope. Forall imaging, about 256 by 256 pixels can be acquired for one frame withan about 200 μs of pixel dwell time (e.g., 13 s per frame) for laserscanning and about 100 μs of time constant (e.g., 6 db filter) from thelock-in amplifier. For FIGS. 41 a-41 f and 4-color pr-SRS imaging atchannels 1606 cm-1 and 1626 cm-1, laser powers can be Ppump=24 mW,Pstokes=48 mW. For FIGS. 41 g and 41 h , laser powers can be Ppump=24mW, Pstokes=12 mW. For FIGS. 41 i and 41 j , laser power can be 24 mWfor two-photon imaging. For 4-color pr-SRS imaging at channels 1642cm-1, 1657 cm-1 and 2940 cm-1 (e.g., cellular background) laser powerscan be Ppump=24 mW, Pstokes=48 mW.

Exemplary SRS Spectra

Stimulated Raman scattering spectra for all fluorophores can be acquiredby fixing the stokes beam laser at about 1064.2 nm, and scanning thepump laser through a designated wavelength range point by point.

Exemplary Materials

Fluorophores can include, for example: 5-Ethynyl-2′-deoxyuridine(T511285, Aldrich); L-Azidohomoalanine (“AHA”) (C10102, Invitrogen),Click-iT® Cell Reaction Buffer Kit (C10269, Invitrogen).

Fluorophores for SRS in FIG. 40 a include: Benzotriazole (B11400, Sigma,λmax˜270 nm); Coumarin 153 (546186, Sigma, λmax˜422 nm); Rhodamine 6G(83697 Sigma, λmax˜528 nm); Rhodamine B (83689 Sigma, λmax˜553 nm);Sulforhodamine 101 (S7635 Sigma, λmax˜580 nm); Alexa Fluor® 633 NHSEster (A-20105, Invitrogen, ˜max˜633 nm); CF640R-azide (92085, Biotium,˜max˜640 nm); Cy5-azide (A3030, Lumiprobe, ˜max˜645 nm); sulfo-cy5-azide(777323 Sigma, λmax˜646 nm); Alexa647-azide (A10277, Invitrogen,˜max˜650 nm); Dylight 650 NHS Ester (62265, Thermo Scientific, ˜max˜650nm); Atto 655 azide (11774 Sigma, λmax˜660 nm); Cy5.5-azide (178,AAT-bioquest, ˜max˜678 nm); ATTO680 (94875 Sigma, λmax˜680 nm);Rhodamine 800 (83701 Sigma, λmax˜680 nm); Alexa 680 NHS Ester (A-20008,Invitrogen, λmax˜680 nm); Alexa700 NHS Ester (A-20010, Invitrogen,˜max˜700 nm); ATTO700 (30674 Sigma, λmax˜700 nm); ATTO725 (47156 Sigma,λmax˜725 nm); ATTO740 (91394 Sigma, λmax˜740 nm); Alexa750 NHS Ester(A-20011, Invitrogen); Cy7-azide (A5030, Lumiprobe, λmax˜749 nm);3,3′-Diethylthiatricarbocyanine iodide (381306 Sigma, λmax˜765 nm);Cy7.5-azide (A6030, Lumiprobe, ˜max˜788 nm); IR820 (543365 Sigma,λmax˜820 nm).

Other Fluorophores can include: IR895 (392375 Sigma, λmax˜895 nm);MitoTracker® Orange CMTMRos (M-7510, Invitrogen); NucBlue® Fixed CellReadyProbes® Reagent (“DAPI”) (R37606, Invitrogen).

Primary antibodies can include: Anti-Fibrillarin antibody—NucleolarMarker (ab5821, Abcam); Anti-200 kD Neurofilament Heavy antibody(ab4680, Abcam); Anti-α-Tubulin antibody (T9026, Sigma); Anti-Giantinantibody (ab24586, Abcam); Anti-Tom20 Antibody (sc-11415, Santa CruzBiotechnology); Anti-Calnexin antibody—ER Membrane Marker (ab140818,Abcam).

Secondary antibodies conjugated with fluorophores can include, e.g.:Goat-anti-Rabbit IgG-Atto 740 antibody (49559, Sigma); Goat-anti-MouseIgG-Atto 700 antibody (2110, Hypermol); Goat anti-Chicken IgY DyLight488 antibody (SA5-10070, Thermo Scientific).

Exemplary Sample Preparation for Intracellular Cell Imaging

For immuno-staining cells can be fixed in methanol for about 28 min orfirst in about 4% PFA for about 8 min and then replaced with methanolfor about 20 min more. Cells can then be washed with about 10% goatserum/1% BSA/0.3M glycine solution twice before permealization in about0.01% triton PBS for about 45 min. Primary antibody can then be addedwith about 1:200 dilution in about 3% BSA in 4 C overnight. Afterblocking with about 10% goat serum for about 30 min, secondary antibodyconjugated with fluorophores can be added with about 1:100 dilution inabout 10% goat serum in 4 C overnight.

For 7-color pr-SRS and fluorescence tandem imaging, HeLa cells can beseeded on a coverslip in a petri-dish with about 2 mL of DMEM for about20 h, and then replaced with Methionine-deficient medium for about 30min. Then about 1 mM AHA and about 100 μM EdU can be added in to mediumfor about 18 hr. An about 400 nM MitoTracker® Orange can be added intomedium for about 30 min before fixation of the cell with about 4% PFAfor about 8 min and then replaced with methanol for about 20 min more.Immuno-staining follows the procedure above. After immuno-staining,about 4 μM Cy5.5-azide can be added to the cells with click-it CellReaction Buffer for the reaction with EdU following the manual fromInvitrogen. After washing with PBS, about 4 μM alexa647-alkyne can beadded to the cells with click-it Cell Reaction Buffer for the reactionwith AHA. At last, DAPI can be added to cells for 20 min following theinstruction from manual.

DMEM was made of about 90% DMEM medium (e.g., 11965, invitrogen), about10% FBS (e.g., 10082, invitrogen) and about 1× penicillin/streptomycin(e.g., 15140, invitrogen); Methionine-deficient medium was made bysupplying about 4 mM L-glutamine, about 0.2 mM L-cystine, about 10% FBSand about 1% penicillin/streptomycin to the DMEM medium withoutL-methionine, L-cysteine and L-glutamine.

Exemplary Linear Combination Procedure

Because the exemplary SRS signal can be linearly dependent on theanalyte concentration, the 4-channel pr-SRS signal for theconcentrations of the ATTO740 labeled nucleoli Fibrillarin protein,ATTO700 labeled α-tubulin, Cy5.5 labeled EdU and Alexa647 labeled AHAcan subtract the cellular background contribution calibrated from 2940cm-1 channel and can be expressed in the following the exemplary matrix:

${{\begin{bmatrix}{{cross} - {section}} \\{matrix}\end{bmatrix}\begin{bmatrix}C_{nucleoli} \\C_{tubulin} \\C_{Edu} \\C_{AHA}\end{bmatrix}} = \begin{bmatrix}{S_{1657} - \left( {S_{2940}/1.63} \right)} \\{S_{1626} - \left( {S_{2940}/2.45} \right)} \\{S_{1642} - \left( {S_{2940}/556} \right)} \\{S_{1606} - \left( {S_{2940}/11.46} \right)}\end{bmatrix}},{and}$ $\begin{bmatrix}{{cross} - {section}} \\{matrix}\end{bmatrix} = \begin{bmatrix}\sigma_{{{ATTO}740},{1657}} & \sigma_{{{ATTO}700},{1657}} & \sigma_{{{Cy}55},{1657}} & \sigma_{{{alexa}647},{1657}} \\\sigma_{{{ATTO}740},{1642}} & \sigma_{{{ATTO}700},{1642}} & \sigma_{{{Cy}55},{1642}} & \sigma_{{{alexa}647},{1642}} \\\sigma_{{{ATTO}740},{1626}} & \sigma_{{{ATTO}700},{1626}} & \sigma_{{{Cy}55},{1626}} & \sigma_{{{alexa}647},{1626}} \\\sigma_{{{ATTO}740},{1606}} & \sigma_{{{ATTO}700},{1606}} & \sigma_{{{Cy}55},{1696}} & \sigma_{{{alexa}647},{1606}}\end{bmatrix}$

Therefore, concentration of each labeled molecules can be solved by:

$\begin{bmatrix}C_{nucleoli} \\C_{tubulin} \\C_{Edu} \\C_{AHA}\end{bmatrix} = {\begin{bmatrix}{{cross} - {section}} \\{matrix}\end{bmatrix}^{- 1}\begin{bmatrix}{S_{1657} - \left( {S_{2940}/1.63} \right)} \\{S_{1626} - \left( {S_{2940}/2.45} \right)} \\{S_{1642} - \left( {S_{2940}/556} \right)} \\{S_{1606} - \left( {S_{2940}/11.46} \right)}\end{bmatrix}}$

Each fluorophore cross section number can be measured using about a 500μM solution in each channel by SRS under the same power and acquisitiontime as in final the cellular imaging condition. Therefore, the solvedmolecule concentrations can be in the unit(s) of μM.

FIG. 44 illustrates exemplary raw images for 4-color pr-SRS microscopyat channels of 1606 cm-1, 1626 cm-1, 1642 cm-1, 1657 cm-1 and 2940 cm-1before linear combination algorithm retrieval.

The foregoing merely illustrates the principles of the disclosure.Various modifications and alterations to the described exemplaryembodiments will be apparent to those skilled in the art in view of theteachings herein. It will thus be appreciated that those skilled in theart will be able to devise numerous systems, arrangements, andprocedures which, although not explicitly shown or described herein,embody the principles of the disclosure and can be thus within thespirit and scope of the disclosure. Various different exemplaryembodiments can be used together with one another, as well asinterchangeably therewith, as should be understood by those havingordinary skill in the art. In addition, certain terms used in thepresent disclosure, including the specification, drawings and claimsthereof, can be used synonymously in certain instances, including, butnot limited to, e.g., data and information. It should be understoodthat, while these words, and/or other words that can be synonymous toone another, can be used synonymously herein, that there can beinstances when such words can be intended to not be used synonymously.Further, to the extent that the prior art knowledge has not beenexplicitly incorporated by reference herein above, it is explicitlyincorporated herein in its entirety. All publications referenced areincorporated herein by reference in their entireties.

EXEMPLARY REFERENCES

The following references are hereby incorporated by reference in theirentirety.

-   1. Chalfie M, Tu Y, Euskirchen G, Ward W W P D (1994) Green    fluorescent protein as a marker gene expression. Science    263:802-804.-   2. Tsien R Y (1998) The Green Fluorescent. Annu. Rev. Biochemistry    67:509-544.-   3. Resch-Genger U, Grabolle M, Cavaliere-Jaricot S, Nitschke R N    T (2008) Quantum dots versus organic dyes as fluorescent labels.    Nature methods 5:763-775.-   4. Miyawaki A, Sawano A, Kogure T (2003) Lighting up cells:    labelling proteins with fluorophores. Nature cell biology.-   5. Knoll B, Keilmann F (1999) Near-field probing of vibrational    absorption for chemical microscopy. Nature 399:7-10.-   6. Turrell G, Corset J (1996) raman microscopy developments and    application-   7. Evans C L, Xie X S (2008) Coherent anti-stokes Raman scattering    microscopy: chemical imaging for biology and medicine. Annual review    of analytical chemistry (Palo Alto, Calif.) 1:883-909.-   8. Freudiger C, Min W, Saar B, Lu S, Holtom G (2008) Label-free    biomedical imaging with high sensitivity by stimulated Raman    scattering microscopy. Science 322:1857-1861.-   9. Min W, Freudiger C W, Lu S, Xie X S (2011) Coherent nonlinear    optical imaging: beyond fluorescence microscopy. Annual review of    physical chemistry 62:507-30.-   10. Prescher J a, Bertozzi C R (2005) Chemistry in living systems.    Nature chemical biology 1:13-21.-   11. Sletten E M, Bertozzi C R (2009) Bioorthogonal chemistry:    fishing for selectivity in a sea of functionality. Angewandte Chemie    (International ed. in English) 48:6974-98.-   12. Lim R K V, Lin Q (2010) Bioorthogonal chemistry: recent progress    and future directions. Chemical communications (Cambridge, England)    46:1589-600.-   13. Yamakoshi H et al. (2011) Imaging of EdU, an alkyne-tagged cell    proliferation probe, by Raman microscopy. Journal of the American    Chemical Society 133:6102-5.-   14. Yamakoshi H et al. (2012) Alkyne-tag Raman imaging for    visualization of mobile small molecules in live cells. Journal of    the American Chemical Society 134:20681-9.-   15. Bloembergen N (1967) The Stimulated Raman Effect. American    Journal of Physics 35:989-1023.-   16. Masters B R, So PTC, Mantulin W W (2008) Handbook of biomedical    nonlinear optical microscopy. eds Masters B R, So PTC (Oxford    University Press)-   17. Saar B G et al. (2010) Video-rate molecular imaging in vivo with    stimulated Raman scattering. Science (New York, N.Y.) 330:1368-70.-   18. Salic A, Mitchison T J (2008) A chemical method for fast and    sensitive detection of DNA synthesis in vivo. Proceedings of the    National Academy of Sciences of the United States of America    105:2415-20.-   19. Neef A B, Luedtke N W (2011) Dynamic metabolic labeling of DNA    in vivo with arabinosyl nucleosides. Proceedings of the National    Academy of Sciences of the United States of America 108:20404-9.-   20. Jao C Y, Salic A (2008) Exploring RNA transcription and turnover    in vivo by using click chemistry. Proceedings of the National    Academy of Sciences of the United States of America 105:15779-84.-   21. Beatty K E et al. (2006) Fluorescence visualization of newly    synthesized proteins in mammalian cells. Angewandte Chemie    (International ed. in English) 45:7364-7.-   22. Liu J, Xu Y, Stoleru D, Salic A (2012) Imaging protein synthesis    in cells and tissues with an alkyne analog of puromycin. Proceedings    of the National Academy of Sciences of the United States of America    109:413-8.-   23. Jao C Y, Roth M, Welti R, Salic A (2009) Metabolic labeling and    direct imaging of choline phospholipids in vivo. Proceedings of the    National Academy of Sciences of the United States of America    106:15332-7.-   24. Hershey JWB, Sonenberg N, Mathews M B Eds. (2012) Protein    synthesis and translational control. Cold Spring Harbor Laboratory    Press.-   25. Martin K C, Barad M, Kandel E R (2000) Local protein synthesis    and its role in synapse-specific plasticity. Curr. Opin. Neurobiol.    10:587-592.-   26. Kandel E R (2001) The molecular biology of memory storage: a    dialogue between genes and synapses. Science 294:1030-1038.-   27. Ho V M, Lee J A, Martin K C (2011) The cell biology of synaptic    plasticity. Science 334:623-628.-   28. Chalfie M, Tu Y, Euskirchen G, Ward W W, Prasher D C (1994)    Green fluorescent protein as a marker for gene expression. Science    263:802-805.-   29. Tsien R Y (1998) The green fluorescent protein. Annu. Rev.    Biochem. 67:509-544.-   30. Dieterich D C, Link A J, Graumann J, Tirrell D A, Schuman E    M (2006) Selective identification of newly synthesized proteins in    mammalian cells using bioorthogonal noncanonical amino acid tagging    (BONCAT). Proc. Natl. Acad. Sci. USA 103:9482-9487.-   31. Beatty K E et al. (2006) Fluorescence visualization of newly    synthesized proteins inmammalian cells. Angew. Chem. 45:7364-7367.-   32. Beatty K E, Tirrell D A (2008) Two-color labeling of temporally    defined proteinpopulations in mammalian cells. Bioorg. Med. Chem.    Lett. 18:5995-5999.-   33. Roche F K, Marsick B M, Letourneau P C (2009) Protein synthesis    in distal axons is notrequired for growth cone responses to guidance    cues. J Neurosci. 29:638-652.-   34. Dieterich D C et al. (2010) In situ visualization and dynamics    of newly synthesizedproteins in rat hippocampal neurons. Nat.    Neurosci. 13:897-905.-   35. Tcherkezian J, Brittis P A, Thomas F, Roux P P, Flanagan J    G (2010) Transmembrane receptor DCC associates with protein    synthesis machinery and regulates translation. Cell 141:632-644.-   36. Hinz F I, Dieterich D C, Tirrell D A, Schuman E M (2012)    Non-canonical amino acid labeling in vivo to visualize and affinity    purify newly synthesized proteins in larval zebrafish. ACS Chem.    Neurosci. 3:40-49.-   37. Liu J, Xu Y, Stoleru D, Salic A (2012) Imaging protein synthesis    in cells and tissues with an alkyne analog of puromycin. Proc. Natl.    Acad. Sci. USA 109:413-418.-   38. Boyce M, Bertozzi C R (2011) Bringing chemistry to life. Nat.    Methods 8:638-642.-   39. Schoenheimer R, Rittenberg D (1936) Deuterium as an indicator in    the study of intermediary metabolism. J. Biol. Chem. 111:163-168.-   40. Schoenheimer R, Rittenberg D (1938) Application of isotopes to    the study of intermediary metabolism. Science 87:221-226.-   41. Ong S E et al. (2002) Stable isotope labeling by amino acids in    cell culture, SILAC, as a simple and accurate approach to expression    proteomics. Mol. Cell. Proteomics 1:376-386.-   42. Mann M (2006) Functional and quantitative proteomics using    SILAC. Nat. Rev. Mol. Cell. Biol. 7:952-958.-   43. Harsha H C, Molina H, Pandey A (2008) Quantitative proteomics    using stable isotope labeling with amino acids in cell culture. Nat.    Protoc. 3:505-516.-   44. Geiger T et al. (2011) Use of stable isotope labeling by amino    acids in cell culture as a spike-in standard in quantitative    proteomics. Nat. Protoc. 6:147-157.-   45. Ingolia N T, Lareau L F, Weissman J S (2011) Ribosome profiling    of mouse embryonic stem cells reveals the complexity and dynamics of    mammalian proteomes. Cell. 147:789-802.-   46. Potma E O, Xie X S (2008) Theory of spontaneous and coherent    Raman scattering in Handbook of Biomedical Nonlinear Optical    Microscopy; Masters B R, So PTC Eds. Oxford University Press: New    York, N.Y., USA.-   47. Zumbusch A, Holtom G R, Xie X S (1999) Three-dimensional    vibrational imaging by coherent anti-Stokes Raman scattering. Phys.    Rev. Lett. 82:4142-4145.-   48. Evans C L, Xie X S (2008) Coherent anti-Stokes Raman scattering    microscopy: chemical imaging for biology and medicine. Annu. Rev.    Anal. Chem. 1:883-909.-   49. Cheng J X, Xie X S (2004) Coherent anti-Stokes Raman scattering    microscopy: instrumentation, theory, and applications. J. Phys.    Chem. B 108:827-840.-   50. Pezacki J P et al. (2011) Chemical contrast for imaging living    systems: molecular vibrations drive CARS microscopy. Nat. Chem.    Biol. 7:137-145.-   51. Suhalim J L, Boik J C, Tromberg B J, Potma E O (2012) The need    for speed. J. Biophotonics 5:387-95.-   52. Ploetz E, Laimgruber S, Berner S, Zinth W, Gilch P (2007)    Femtosecond stimulated Raman microscopy. Appl. Phys. B 87:389-393.-   53. Freudiger C W et al. (2008) Label-free biomedical imaging with    high sensitivity by stimulated Raman scattering microscopy. Science    322:1857-1861.-   54. Ozeki Y, Dake F, Kajiyama S, Fukui K, Itoh K (2009) Analysis and    experimental assessment of the sensitivity of stimulated Raman    scattering microscopy. Opt. express. 17:3651-3658.-   55. Nandakumar P, Kovalev A, Volkmer A (2009) Vibrational imaging    based on stimulated Raman scattering microscopy. New J. Phys.    11:033026.-   56. Saar B G et al. (2010) Video-rate molecular imaging in vivo with    stimulated Raman scattering. Science 330:1368-1370.-   57. Zhang D, Slipchenko M N, Cheng J X (2011) Highly sensitive    vibrational imaging by femtosecond pulse Stimulated raman Loss. J.    Phys. Chem. Lett. 2:1248-1253.-   58. Wang M C, Min W, Freudiger C W, Ruvkun G, Xie X S (2011) RNAi    screening for fat regulatory genes with SRS microscopy. Nat. methods    8:135-138.-   59. Zhang X et al. (2012) Label-free live-cell imaging of nucleic    acids using stimulated Raman scattering microscopy. Chemphyschem.    13:1054-1059.-   60. Fu D et al. (2012) Quantitative chemical imaging with multiplex    stimulated Raman scattering microscopy. J. Am. Chem. Soc. 134:    3623-3626.-   61. Ozeki Y et al. (2012) High-speed molecular spectral imaging of    tissue with stimulated Raman scattering. Nature Photon. 6:845-851.-   62. Einstein A (1917) On the quantum theory of radiation. Phys. Z.    18:121-128.-   63. Bloembergen N (1967) The Stimulated Raman Effect. Am. J. Phys.    35:989-1023.-   64. Min W, Freudiger C W, Lu S, Xie X S (2011) Coherent nonlinear    optical imaging: beyond fluorescence microscopy. Annu Rev Phys Chem.    62:507-530.-   65. Min W (2011) Label-free optical imaging of nonfluorescent    molecules by stimulated radiation. Curr. Opin. Chem. Biol.    15:831-837.-   66. Okayasu T, Ikeda M, Akimoto K, Sorimachi K (1997) The amino acid    composition of mammalian and bacterial cells. Amino Acids    13:379-391.-   67. Phair R D and Misteli T (2000) High mobility of proteins in the    mammalian cell nucleus. Nature 404: 604-609.-   68. Andersen J S et al. (2005) Nucleolar proteome dynamics. Nature    433:77-83.-   69. Boisvert F M et al. (2012) A quantitative spatial proteomics    analysis of proteome turnover in human cells. Mol. Cell. Proteomics.    11(3): M111.011429.-   70. Piez K A and Eagle H (1958) The free amino acid pool of cultured    human cells. J. Biol. Chem. 231: 533-545-   71. Lechene C P, Luyten Y, McMahon G, Distel D L (2007) Quantitative    imaging of nitrogen fixation by individual bacteria within animal    cells. Science 317:1563-1566.-   72. Zhang D S et al. (2012) Multi-isotope imaging mass spectrometry    reveals slow protein turnover in hair-cell stereocilia. Nature    481:520-524.-   73. van Manen H J, Lenferink A, Otto C (2008) Noninvasive imaging of    protein metabolic labeling in single human cells using stable    isotopes and Raman microscopy. Anal. chem. 80:9576-9582.-   74. Ji Minbiao et al. (2013) Rapid, label-free detection of brain    tumors with stimulated Raman scattering miscroscopy. Sci. Transl.    Med. 5(201): 201ra119.-   76. Saar et al. (2011) Imaging drug delivery to skin with stimulated    Raman scattering microscopy. Mol. Pharm. 8(3): 969-75.-   77. Cui et al. (2009) Comparing coherent and spontaneous Raman    scattering under biological imaging conditions. Opt. Lett. 34(16):    773-775.-   78. Petrov et al. (2007) Comparision of coherent and spontaneous    Raman microspectroscopies for noninvasive detection of single    bacterial endospores. Proc. Natl. Acad. Sci. USA. 104(19): 7776-9.-   79. Nie, S., Chiu, D. T. & Zare, R. N. Probing individual molecules    with confocal fluorescence microscopy. Science 266, 1018-1021    (1994).-   80. Moerner, W. E. & Orrit, M. Illuminating single molecules in    condensed matter. Science 283, 1670-1676 (1999).-   81. Yuste, R. Fluorescence microscopy today. Nat. Methods 2, 902-904    (2005).-   82. Denk, W., Strickler, J. H. & Webb, W. W. Two-photon laser    scanning fluorescence microscopy. Science 248, 73-76 (1990).-   83. Hell, S. W. & Wichmann, J. Breaking the diffraction resolution    limit by stimulated emission: stimulated-emission-depletion    fluorescence microscopy. Opt. Lett. 19, 780-2 (1994).-   84. Betzig, E. et al. Imaging intracellular fluorescent proteins at    nanometer resolution. Science 313, 1642-1645 (2006).-   85. Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit    imaging by stochastic optical reconstruction microscopy (STORM).    Nat. Methods 3, 793-795 (2006).-   86. Ha, T. et al. Probing the interaction between two single    molecules: fluorescence resonance energy transfer between a single    donor and a single acceptor. Proc. Natl. Acad. Sci. U.S.A 93,    6264-6268 (1996).-   87. Myers, A B. Molecular electronic spectral broadening in liquids    and glasses. Annu. Rev. Phys. Chem. 49, 267-295 (1998).-   88. Nemkovich, N., Rubinov, A. & Tomin, V. Inhomogeneous Broadening    of Electronic Spectra of Dye Molecules in Solutions. Top. Fluoresc.    Spectrosc. S E—8 2, 367-428 (2002).-   89. Gaiduk, A., Yorulmaz, M., Ruijgrok, P. V & Orrit, M.    Room-temperature detection of a single molecule's absorption by    photothermal contrast. Science 330, 353-356 (2010).-   90. Chong, S., Min, W. & Xie, X. S. Ground-state depletion    microscopy: Detection sensitivity of single-molecule optical    absorption at room temperature. J. Phys. Chem. Lett. 1, 3316-3322    (2010).-   91. Kukura, P., Celebrano, M., Renn, A. & Sandoghdar, V.    Single-molecule sensitivity in optical absorption at room    temperature. J. Phys. Chem. Lett. 1, 3323-3327 (2010).-   92. Giesen, C. et al. Highly multiplexed imaging of tumor tissues    with subcellular resolution by mass cytometry. Nat. Methods 11,    417-22 (2014).-   93. Angelo, M. et al. Multiplexed ion beam imaging of human breast    tumors. Nat. Med. 20, 436-42 (2014).-   94. Barlogie, B. et al. Flow Cytometry in Clinical Cancer Research    Flow Cytometry in Clinical Cancer Researchl. 43, 3982-3997 (1983).-   95. Geiger, B., Spatz, J. P. & Bershadsky, A. D. Environmental    sensing through focal adhesions. Nat. Rev. Mol. Cell Biol. 10, 21-33    (2009).-   96. Cheng, J.-X. & Xie, X. S. Coherent Raman Scattering Microscopy.    CRC press (2012).-   97. Nie, S. Probing Single Molecules and Single Nanoparticles by    Surface-Enhanced Raman Scattering. Science. 275, 1102-1106 (1997).-   98. Kneipp, K. et al. Single molecule detection using    surface-enhanced Raman scattering (SERS). Phys. Rev. Lett. 78,    1667-1670 (1997).-   99. Freudiger, C., Min, W., Saar, B., Lu, S. & Holtom, G. Label-free    biomedical imaging with high sensitivity by stimulated Raman    scattering microscopy. Science (80-.). 322, 1857-1861 (2008).-   100. Min, W., Freudiger, C. W., Lu, S. & Xie, X. S. Coherent    nonlinear optical imaging: beyond fluorescence microscopy. Annu.    Rev. Phys. Chem. 62, 507-530 (2011).-   101. Saar, B. G. et al. Video-rate molecular imaging in vivo with    stimulated Raman scattering. Science 330, 1368-1370 (2010).-   102. Ji, M. et al. Rapid, label-free detection of brain tumors with    stimulated Raman scattering microscopy. Sci. Transl. Med. 5,    201ra119 (2013).-   103. Wei, L. et al. Live-cell imaging of alkyne-tagged small    biomolecules by stimulated Raman scattering. Nat. Methods 11, 410-2    (2014).-   104. Yamakoshi, H. et al. Alkyne-tag Raman imaging for visualization    of mobile small molecules in live cells. J. Am. Chem. Soc. 134,    20681-20689 (2012).-   105. Results, E. Probe-frequency dependence of the resonant Inverse    Raman band shape. Oct. 89, 3945-3950 (1988).-   106. McCamant, D. W., Kukura, P. & Mathies, R. A. Femtosecond    Broadband Stimulated Raman: A New Approach for High-Performance    Vibrational Spectroscopy. Appl. Spectrosc. 57, 1317-1323 (2003).-   107. Kim, H. M., Kim, H., Yang, I., Jin, S. M. & Suh, Y. D.    Time-gated pre-resonant femtosecond stimulated Raman spectroscopy of    diethylthiatricarbocyanine iodide. Phys. Chem. Chem. Phys. 16,    5312-8 (2014).-   108. Asher, S. A. U V resonance Raman studies of molecular structure    and dynamics: applications in physical and biophysical chemistry.    Annu. Rev. Phys. Chem. 39, 537-588 (1988).

1. A method for imaging a living cell or a living organism, comprising:introducing an effective amount of a bond-edited compound into a livecell or a living organism, wherein the bond-edited compound comprises avibrational tag; and detecting the vibrational tag in the cell or theorganism with stimulated Raman scattering (SRS) imaging. 2-44.(canceled)